Detection of susceptibility to autoimmune diseases

ABSTRACT

The present invention provides methods and reagents for determining sequence variants present at the IL4 receptor, IL-4 and IL-13 loci, which facilitate identifying individuals at risk for type 1 diabetes.

The present patent application is a continuation of U.S. patentapplication Ser. No. 10/267,844 filed Oct. 8, 2002, which is acontinuation-in-part of U.S. patent application Ser. No. 10/264,965filed Oct. 4, 2002, which is a continuation-in-art of U.S. patentapplication Ser. No. 10/189,956 filed Jul. 3, 2002, which claimspriority to U.S. provisional patent application Ser. No. 60/306,912filed Jul. 20, 2001, all of which are herein incorporated by referencein their entireties.

1. FIELD OF THE INVENTION

The present invention relates to the fields of immunology and molecularbiology. In particular, it relates to methods and reagents for detectingan individual's risk for autoimmune diseases. More specifically, itrelates to methods and reagents for detecting an individual's increasedor decreased risk for type 1 diabetes.

2. DESCRIPTION OF RELATED ART

The immunological response to an antigen is mediated through theselective differentiation of CD4+ T helper precursor cells (Th0) to Thelper type 1 (Th1) or T helper type 2 (Th2) effector cells, withfunctionally distinct patterns of cytokine (also described aslymphokine) secretion. Th1 cells secrete interleukin 2 (IL-2), IL-12,tumor necrosis factor (TNF), lymphotoxin (LT), and interferon gamma(IFN-γ) upon activation, and are primarily responsible for cell-mediatedimmunity such as delayed-type hypersensitivity. Th2 cells secrete IL-4,IL-5, IL-6, IL-9, and IL-1 3 upon activation, and are primarilyresponsible for extracellular defense mechanisms. The role of Th1 andTh2 cells is reviewed in Peltz, 1991, Immunological Reviews, 123: 23-35,incorporated herein by reference.

IL4 and IL13 play a central role in IgE-dependent inflammatoryreactions. IL4 induces IgE antibody production by B Cells and furtherprovides a regulatory function in the differentiation of Th0 to Th1 orTh2 effector cells by both promoting differentiation into Th2 cells andinhibiting differentiation into Th1 cells. IL13 also induces IgEantibody production by B Cells.

IL4 and IL13 operate through the IL4 receptor (“IL4R”), found on both Band T cells, and the IL13R, found on B cells, respectively. The humanIL4 receptor (IL4R) is a heterodimer comprising the IL4R α chain and theIL2 receptor γ chain. The α-chain of the IL4 receptor also serves as theα-chain of the IL13 receptor. IL4 binds to both IL4R and IL13R throughthe IL4R α-chain and can activate both B and T cells, whereas IL13 bindsonly to IL13R through the IL13R α1 chain and activates only T cells.

3. SUMMARY OF INVENTION

The present invention provides methods for detecting an individual'sincreased or decreased risk for an autoimmune disease such as type 1diabetes, also known as insulin-dependent diabetes mellitus (“IDDM”).The present invention also provides kits, reagents and arrays useful fordetecting an individual's risk for autoimmune diseases such as type 1diabetes.

In one aspect, the present invention provides a method for detecting anindividual's increased or decreased risk for an autoimmune disease suchas type 1 diabetes by detecting the presence of a type 1diabetes-associated polymorphism in the IL4R, IL4 or IL13 loci in anucleic acid sample of the individual, wherein the presence of saidpolymorphism indicates the individual's increased risk for type 1diabetes.

In one aspect, the present invention provides a method for detecting anindividual's increased or decreased risk for an autoimmune disease suchas type 1 diabetes by detecting the presence of a type 1diabetes-associated polymorphism in the IL4R, IL4 or IL13 loci in anucleic acid sample of the individual, wherein the presence of saidpolymorphism indicates the individual's increased risk for type 1diabetes.

In one embodiment, the polymorphism is an IL4R polymorphism. In anotherembodiment, the polymorphism is an IL4 polymorphism. In anotherembodiment, the polymorphism is an IL13 polymorphism. In anotherembodiment, an IL4R polymorphism and an IL4 polymorphism are detected.In another embodiment, an IL4R polymorphism and an IL13 polymorphism aredetected. In another embodiment, an IL4 polymorphism and an IL13polymorphism are detected. In another embodiment, an IL4R polymorphism,an IL4 polymorphism and an IL13 polymorphism are detected.

In another embodiment, the IL4R polymorphism is selected from the IL4Rpolymorphisms listed in Table 21. In another embodiment, the IL4polymorphism is the the IL4(−524) polymorphisms listed in Table 21. Inanother embodiment, the IL13 polymorphism is selected from the IL4Rpolymorphisms listed in Table 21. In another embodiment, 2 or more IL4Rpolymorphisms selected from the IL4R polymorphisms listed in Table 21are detected. In another embodiment, 6 or more IL4R polymorphismsselected from the IL4R polymorphisms listed in Table 21 are detected. Inanother embodiment, 7 or more IL4R polymorphisms selected from the IL4Rpolymorphisms listed in Table 21 are detected. In another embodiment, 8or more IL4R polymorphisms selected from the IL4R polymorphisms listedin Table 21 are detected. In another embodiment, all 10 IL4Rpolymorphisms listed in Table 21 are detected.

The individual can belong to any race or population. In one embodiment,the individual is an Asian, preferably a Filipino, or a Caucasian.

The nucleic acid sample can be obtained from any part of theindividual's body, including, but not limited to hair, skin, nails,tissues or bodily fluids such as saliva, blood, etc. The nucleic acidsample can, but need not, be amplified by any amplification methodincluding, but not limited to, polymerase chain reaction (“TCR”).

The polymorphism can be any predisposing or protective polymorphism inthe IL4R, IL4 or IL13 loci. In one embodiment of the invention, thepolymorphism can be any polymorphism identified as predisposing orprotective by methods taught herein. In one embodiment, the polymorphismcan be a single nucleotide polymorphism (“SNP”) in the IL-4 receptor(“IL4-R”), IL4 or IL13 loci. In another embodiment, specific haplotypesin the IL4R, IL4 and IL13 loci as well as specific combinations of, andinteractions between, SNPs at these loci can be indicative of anincreased or a decreased risk to an autoimmune disease such as type 1diabetes.

The polymorphism can be detected by any method known in the art fordetecting the presence of a specific polymorphism in a nucleic acidsample. These methods include, but are not limited to, contacting thenucleic acid sample with one or more nucleic acid molecules thathybridize under stringent hybridization conditions to at least one type1 diabetes-associated IL4R, IL4 or IL13 polymorphism and detecting thehybridization, detection by amplification of the nucleic acid sample by,for example, PCR, and by direct sequencing of the nucleic acid sample.

Another aspect of the invention relates to a kit useful for detectingthe presence of a predisposing or a protective polymorphism in the IL4R,IL4 or IL13 loci in a nucleic acid sample of an individual whose riskfor type 1 diabetes is being assessed. The kit can comprise one or moreoligonucleotides capable of detecting a predisposing or protectivepolymorphism in the IL4R, IL4 or IL13 loci as well as instructions forusing the kit to detect susceptibility for an autoimmune disease such astype 1 diabetes. In preferred embodiments, the oligonucleotide oroligonucleotides each individually comprise a sequence that hybridizesunder stringent hybridization conditions to at least one type 1diabetes-associated IL4R, IL4 or IL13 polymorphism. In some embodiments,the oligonucleotide or oligonucleotides each individually comprise asequence that is fully complementary to a nucleic acid sequencecomprising a type 1 diabetes-associated IL4R, IL4 or IL13 polymorphism.

In some embodiments, the oligonucleotide can be used to detect thepresence of a type 1 diabetes-associated IL4R, IL4 or IL13 polymorphismby hybridizing to the polymorphism under stringent hybridizingconditions. In some embodiments, the oligonucleotide can be used as anextension primer in either an amplification reaction such as PCR or asequencing reaction, wherein the type 1 diabetes-associated IL4R, IL4 orIL13 polymorphism is detected either by amplification or sequencing.

In certain embodiments, the kit can further comprise amplification orsequencing primers which can, but need not, be sequence-specific. Thekit can also comprise reagents for labeling one or more of theoligonucleotides, or comprise labeled oligonucleotides. Optionally, thekit can comprise reagents to detect the label.

In some embodiments, the kit can comprise one or more oligonucleotidesthat can be used to detect the presence of two or more predisposing orprotective IL4R, IL4 or IL 13 polymorphisms or combinations ofpredisposing polymorphisms, protective polymorphisms or both.

In another aspect, the invention provides an array useful for detectingthe presence of a predisposing or a protective IL4R, IL4 or IL13polymorphism in a nucleic acid sample of an individual whose risk fortype 1 diabetes is being assessed. The array can comprise one or moreoligonucleotides capable of detecting a predisposing or protective IL4R,IL4 or IL13 polymorphism. The oligonucleotides can be immobilized on asubstrate, e.g., a membrane or glass. In preferred embodiments, theoligonucleotide or oligonucleotides each individually comprise asequence that can hybridize under stringent hybridization conditions toa nucleic acid sequence comprising a type 1 diabetes-associated IL4R,IL4 or IL13 polymorphism. In some embodiments, the oligonucleotide oroligonucleotides each individually comprise a sequence that is fullycomplementary to a nucleic acid sequence comprising a type 1diabetes-associated IL4R, IL4 or IL13 polymorphism. The oligonucleotideor oligonucleotides can, but need not, be labeled. In some embodiments,the array can be a microarray.

In some embodiments, the array can comprise one or more oligonucleotidesused to detect the presence of two or more predisposing or protectiveIL4R, IL4 or IL13 polymorphisms or combinations of predisposingpolymorphisms, protective polymorphisms or both.

In certain embodiments, an individual's risk for particular Th1-mediateddiseases is diagnosed from the individual's IL4R, IL4 or IL13 genotype.In a preferred embodiment, the Th1-mediated disease is type 1 diabetes.An individual who has at least one polymorphism statistically associatedwith type 1 diabetes possesses a factor contributing to either anincreased or a decreased risk of a type 1 diabetes as compared to anindividual without the polymorphism. The statistical association ofIL4R, IL4 or IL13 polymorphisms (sequence variants) is shown in theexamples.

The genotype can be determined using any method capable of identifyingnucleotide variation consisting of single nucleotide polymorphic sites.The particular method used is not a critical aspect of the invention. Anumber of suitable methods are described below.

In one embodiment of the invention, genotyping is carried out usingoligonucleotide probes specific to variant IL4R, IL4 or IL13 sequences.Preferably, a region of the IL4R, IL4 or IL13 genes which encompassesone or several polymorphic sites of interest is amplified prior to, orconcurrent with, the hybridization of probes directed to such sites.Probe-based assays for the detection of sequence variants are well knownin the art.

Alternatively, genotyping is carried out using allele-specificamplification or extension reactions, wherein allele-specific primersare used which support primer extension only if the targeted allele ispresent. Typically, an allele-specific primer hybridizes to the IL4R,IL4 or IL13 genes such that the 3′ terminal nucleotide aligns with apolymorphic position. Allele-specific amplification reactions andallele-specific extension reactions are well known in the art.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic of a molecular haplotyping method;

FIG. 2 provides an illustration of epistasis between the IL4R SNPs andIL4 and IL13 SNPs.

5. BRIEF DESCRIPTION OF THE TABLES

Table 1 provides the nucleotide sequence of the coding region of an IL4R(SEQ ID NO: 2);

Table 2 provides IL4R, IL4 and IL13 SNPs useful in the methods of theinvention;

Table 3 provides probes used to identify IL4R polymorphisms (SEQ ID NO:3-19);

Table 4 provides computationally estimated haplotype frequenciescompared between Filipino controls and diabetics (SEQ ID NO: 20-24);

Table 5 provides genotypes of affected and nonaffected individuals;

Table 6 provides single nucleotide polymorphisms detected;

Table 7 provides amplicon primers and lengths (SEQ ID NO: 25-36);

Table 8 provides hybridization probes and titers (SEQ ID NO: 37-53);

Table 9 provides allele frequency of wildtype allele in HBDI founders;

Table 10 provides D′ and A values for pairs of IL4R SNPs;

Table 11A provides results of single locus TDT analysis;

Table 11B provides results of single locus TDT analysis;

Table 12 provides allele-specific PCR primers (SEQ ID NO: 54-62);

Table 13 provides IBD distributions for IL4R haplotypes;

Table 14A provides haplotype transmissions;

Table 14B provides haplotype transmissions;

Table 14C provides haplotype transmissions;

Table 15A provides SNP by SNP allele transmissions;

Table 15B provides SNP by SNP allele transmissions;

Table 16A provides a TDT analysis;

Table 16B provides a TDT analysis;

Table 16C provides a TDT analysis;

Table 17A provides a TDT analysis;

Table 17B provides a TDT analysis;

Table 18 provides allele frequencies in Filipino controls and diabetics;

Table 19 provides estimated haplotype frequencies;

Table 20 provides observed haplotype frequencies;

Table 21 provides allele frequencies in diabetics and controls;

Table 22 provides pairwise linkage disequilibrium values for IL4R SNPs;

Table 23 provides pairwise linkage disequilibrium values for IL4 andIL13 SNPs;

Table 24 provides genotype frequencies in patients and controls;

Table 25A provides IL-4R 7-SNP Haplotypes in Filipino diabetics andcontrols;

Table 25B provides estimated IL4R 10-SNP haplotype frequencies indiabetics and controls;

Table 26 provides estimated IL4 and IL13 5-SNP haplotype frequencies indiabetics and controls;

Table 27 provides correlation between genotype frequencies at IL4R SNPsand five IL4 and IL13 SNPs;

Table 28 provides epistatic interaction between IL4R SNPs and five IL4and IL13 SNPs;

Table 29 provides probes used to identify IL4 and IL13 polymorphisms(SEQ ID NO: 63-68);

Table 30 provides amplicon primers and lengths for an IL4 promoter andIL13 SNPs (SEQ ID NO: 69-74);

Table 31 provides amplicon primers and lengths for IL4R promoter SNPs(SEQ ID NO: 75-80);

Table 32 provides amplicon primers and lengths for IL13 promoter SNPs(SEQ ID NO: 81-86);

6. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods, reagents and kits for detectingan individual's increased or decreased risk for an autoimmune disease.Examples of autoimmune diseases include, but are not limited to,multiple sclerosis, myasthenia gravis, Crohn's disease, ulcerativecolitis, primary biliary cirrhosis, type 1 diabetes mellitus (insulindependent diabetes mellitus or IDDM), Grave's disease, autoimmunehemolytic anemia, pernicious anemia, autoimmune thrombocytopenia,vasculitides such as Wegener's granulomatosis, Behcet's disease,rheumatoid arthritis, systemic lupus erythematosus (lupus), scleroderma,systemic sclerosis, Guillain-Barre syndromes, Hashimoto's thyroiditisspondyloarthropathies such as ankylosing spondylitis, psoriasis,dermatitis herpetiformis, inflammatory bowel diseases, pemphigusvulgaris and vitiligo. In certain preferred embodiments, the autoimmunedisease is type 1 diabetes.

Abbreviations and Terminology:

The term “IL4R gene” or “IL4R locus” refers to the genomic nucleic acidsequence that encodes the alpha sub-unit of the interleukin 4 receptorprotein. The nucleotide sequence of a gene, as used herein, encompassescoding regions, referred to as exons, intervening, non-coding regions,referred to as introns, and upstream or downstream regions. Upstream ordownstream regions can include regions of the gene that are transcribedbut not part of an intron or exon, or regions of the gene that comprise,for example, binding sites for factors that modulate gene transcription.The gene sequence of a Human mRNA for IL4R is provided at GenBankaccession number X52425.1 (SEQ ID NO: 1). The coding region is providedas SEQ ID NO: 2. The genomic sequence for the IL4R gene is included inGenBank accession number AC004525.1 (SEQ ID NO: 88).

The term “IL4 gene” or “IL4 locus” refers to the genomic nucleic acidsequence that encodes the interleukin 4 protein. The nucleotide sequenceof a gene, as used herein, encompasses coding regions, referred to asexons, intervening, non-coding regions, referred to as introns, andupstream or downstream regions. Upstream or downstream regions caninclude regions of the gene that are transcribed but not part of anintron or exon, or regions of the gene that comprise, for example,binding sites for factors that modulate gene transcription. The genomicsequence for the IL4 gene is provided at GenBank accession numberM23442.1 (SEQ ID NO: 89).

The term “IL13 gene” or “IL13 locus” refers to the genomic nucleic acidsequence that encodes the interleukin 13 protein. The nucleotidesequence of a gene, as used herein, encompasses coding regions, referredto as exons, intervening, non-coding regions, referred to as introns,and upstream or downstream regions. Upstream or downstream regions caninclude regions of the gene that are transcribed but not part of anintron or exon, or regions of the gene that comprise, for example,binding sites for factors that modulate gene transcription. The genomicsequence for the IL13 gene is provided at GenBank accession numberU10307.1 (SEQ ID NO: 90).

The term “allele”, as used herein, refers to a sequence variant of thegene. Alleles are identified with respect to one or more polymorphicpositions, with the rest of the gene sequence unspecified. For example,an IL4R allele may be defined by the nucleotide present at a single SNP,or by the nucleotides present at a plurality of SNPs. In certainembodiments of the invention, an IL4R is defined by the genotypes of 6,7, 8 or 10 IL4R SNPs. Examples of such IL4R SNPs are provided in Table2, below.

For convenience, the allele present at the higher or highest frequencyin the population will be referred to as the wild-type allele; lessfrequent allele(s) will be referred to as mutant-allele(s). Thisdesignation of an allele as a mutant is meant solely to distinguish theallele from the wild-type allele and is not meant to indicate a changeor loss of function.

The term “predisposing polymorphism” refers to a polymorphism that ispositively associated with an autoimmune disease such as type 1diabetes. The presence of a predisposing polymorphism in an individualcould be indicative that the individual has an increased risk for thedisease relative to an individual without the polymorphism.

The term “protective polymorphism” refers to a polymorphism that isnegatively associated with an autoimmune disease such as type 1diabetes. The presence of a protective polymorphism in an individualcould be indicative that the individual has a decreased risk for thedisease relative to an individual without the polymorphism.

The terms “polymorphic” and “polymorphism”, as used herein, refer to thecondition in which two or more variants of a specific genomic sequence,or the encoded amino acid sequence, can be found in a population. Theterms refer either to the nucleic acid sequence or the encoded aminoacid sequence; the use will be clear from the context. The polymorphicregion or polymorphic site refers to a region of the nucleic acid wherethe nucleotide difference that distinguishes the variants occurs, or,for amino acid sequences, a region of the amino acid where the aminoacid difference that distinguishes the protein variants occurs. As usedherein, a “single nucleotide polymorphism”, or SNP, refers to apolymorphic site consisting of a single nucleotide position.

“Odds Ratio” (“OR”) refers to the ratio of the odds of the disease forindividuals with the marker (allele or polymorphism) relative to theodds of the disease in individuals without the marker (allele orpolymorphism).

“Linkage Disequilibrium” (“LD”) refers to alleles at different loci thatare not associated at random, i.e., not associated in proportion totheir frequencies. If the alleles are in positive linkagedisequilibrium, then the alleles occur together more often than expectedassuming statistical independence. Conversely, if the alleles are innegative linkage disequilibrium, then the alleles occur together lessoften than expected assuming statistical independence.

The term “genotype” refers to a description of the alleles of a gene orgenes contained in an individual or a sample. As used herein, nodistinction is made between the genotype of an individual and thegenotype of a sample originating from the individual. Although,typically, a genotype is determined from samples of diploid cells, agenotype can be determined from a sample of haploid cells, such as asperm cell.

The term “haplotype” refers to a description of the variants of a geneor genes contained on a single chromosome, i.e., the genotype of asingle chromosome. A haplotype is a set of maternally inherited alleles,or a set of paternally inherited alleles, at any locus.

The term “target region” refers to a region of a nucleic acid which isto be analyzed and usually includes at least one polymorphic region.

Individual amino acids in a sequence are represented herein as AN or NA,wherein A is the amino acid in the sequence and N is the position in thesequence. In the case that position N is polymorphic, it is convenientto designate the more frequent variant as A₁N and the less frequentvariant as NA₂. Alternatively, the polymorphic site, N, is representedas A₁NA₂, wherein A₁ is the amino acid in the more common variant and A₂is the amino acid in the less common variant. Either the one-letter orthree-letter codes are used for designating amino acids (see Lehninger,BioChemistry 2nd ed., 1975, Worth Publishers, Inc. New York, N.Y.: pages73-75, incorporated herein by reference). For example, 150V represents asingle-amino-acid polymorphism at amino acid position 50, whereinisoleucine is the present in the more frequent protein variant in thepopulation and valine is present in the less frequent variant. The aminoacid positions are numbered based on the sequence of the mature IL4Rprotein, as described below.

“Stringent” as used herein refers to hybridization and wash conditionsat 50° C. or higher. Other stringent hybridization conditions may alsobe selected. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (Tm) for the specific sequenceat a defined ionic strength and pH. The Tm is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Typically, stringent conditionswill be those in which the salt concentration is at least about 0.02molar at pH 7 and the temperature is at least about 50° C. As otherfactors may significantly affect the stringency of hybridization,including, among others, base composition, length of the nucleic acidstrands, the presence of organic solvents, the extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one.

Representations of nucleotides and single nucleotide changes in DNAsequences are analogous. For example, A398G represents a singlenucleotide polymorphism at nucleotide position 398, wherein adenine isthe present in the more frequent (wild-type) allele in the populationand guanine is present in the less frequent (mutant) allele. Thenucleotide positions are numbered based on the IL4R coding regionsequence provided as SEQ ID NO:2, shown below. It will be clear that ina double stranded form, the complementary strand of each allele willcontain the complementary base at the polymorphic position.

Conventional techniques of molecular biology and nucleic acid chemistry,which are within the skill of the art, are fully explained in theliterature. See, for example, Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins. eds., 1984); the series,Methods in Enzymology (Academic Press, Inc.); and the series, CurrentProtocols in Human Genetics (Dracopoli et al., eds., 1984 with quarterlyupdates, John Wiley & Sons, Inc.); all of which are incorporated hereinby reference. All patents, patent applications, and publicationsmentioned herein, both supra and infra, are incorporated herein byreference.

Association with Type 1 Diabetes

As IL4R, IL4 or IL13 are a small component of the complex system ofgenes involved in an immune response, the effect of the IL4R, IL4 orIL13 loci is expected to be small. Other factors, such as anindividual's HLA genotype, may exert dominating effects which, in somecases, may mask the effect of the IL4R, IL4 or IL13 genotypes. Forexample, particular HLA genotypes are known to have a major effect onthe likelihood of type 1 diabetes (see Noble et al., 1996, Am. J. Hum.Genet, 59:1134-1148, incorporated herein by reference). The IL4R, IL4 orIL13 genotypes are likely to be more informative as an indicator ofpredisposition towards type 1 diabetes among individuals who have HLAgenotypes that confer neither increased nor decreased risk. Furthermore,because allele frequencies at other loci relevant to immunesystem-related diseases differ between populations and, thus,populations exhibit different risks for immune system-related diseases,it is expected that the effect of the IL4R, IL4 or IL13 genotypes may beof different magnitude in some populations. Although the contribution ofthe IL4R, IL4 or IL13 genotypes may be relatively minor by itself,genotyping at the IL4R, IL4 or IL13 loci will contribute informationthat is, nevertheless, useful for a characterization of an individual'spredisposition towards type 1 diabetes. The IL4R, IL4 or IL13 genotypeinformation may be particularly useful when combined with genotypeinformation from other loci.

Methods for Detecting Risk for Autoimmune Diseases

The present invention provides methods of determining an individual'srisk for any autoimmune disease or condition or any Th-1 mediateddisease. Such diseases or conditions include, but are not limited to,multiple sclerosis, myasthenia gravis, Crohn's disease, ulcerativecolitis, primary biliary cirrhosis, type 1 diabetes mellitus (insulindependent diabetes mellitus or IDDM), Grave's disease, autoimmunehemolytic anemia, pernicious anemia, autoimmune thrombocytopenia,vasculitides such as Wegener's granulomatosis, Behcet's disease,rheumatoid arthritis, systemic lupus erythematosus (lupus), scleroderma,systemic sclerosis, Gullian-Barre syndromes, Hashimoto's thyroiditisspondyloarthropathies such as ankylosing spondylitis, psoriasis,dermatitis herpetiformis, inflammatory bowel diseases, pemphigusvulgaris and vitiligo. In certain embodiments of the invention, themethods are used to determine an individual's risk for type 1 diabetes.Preferably, the individual is a human.

Nucleic Acids

Accordingly, one embodiment of the invention is an isolated nucleic acidmolecule comprising a portion of the IL4R, IL4 or IL13 genes, theircomplements, or variants thereof. Preferably said variant comprises atleast one of the polymorphisms identified herein. Even more preferably,said variant comprises at least one of the polymorphisms identifiedherein to be associated with type 1 diabetes. Thus, in one embodiment,the nucleic acid molecule comprises at least one of the IL4R, IL4,and/or IL13 polymorphisms provided in Table 2. In a further embodiment,the nucleic acid molecule comprises or consists of primers and probesspecific to polymorphisms identified in the IL4R, IL4, or IL13 gene,including but not limited to SEQ ID NOS: 3-19, 25-36, 37-53, 54-62,69-74, 75-80, and 81-86.

The isolated nucleic acid molecules may be RNA, mRNA, DNA, cDNA, and maybe double- or single-stranded. They may encode the sense strand, thenon-coding regions, or the antisense strand. The nucleic acid moleculecan include all or a portion of the coding sequence of the gene and canfurther comprise additional non-coding regions such as introns andnon-coding 3′ and 5′ sequences (including regulatory sequences forexample). Additionally, the nucleic acid molecule can be fused to amarker sequence, for example, a sequence that encodes a polypeptide toassist in isolation or purification of the polypeptide.

An “isolated” nucleic acid molecule, as used herein, is one that isseparated from nucleotide sequences which normally flank the nucleicacid molecule and/or has been completely or partially purified fromother biological material (e.g., protein) normally associated with thenucleic acid.

The nucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated. Thus, recombinant DNAcontained in a vector is included in the definition of “isolated” asused herein. Also, isolated nucleic acid molecules include recombinantDNA molecules in heterologous host cells, as well as partially orsubstantially purified DNA molecules in solution. “Isolated” nucleicacid molecules also encompass in vivo and in vitro RNA transcripts ofthe DNA molecules of the present invention. An isolated nucleic acidmolecule or nucleotide sequence can include a nucleic acid molecule ornucleotide sequence that is synthesized chemically or by recombinantmeans. Also, isolated polynucleotides include recombinant DNA moleculesin heterologous organisms, as well as partially or substantiallypurified DNA molecules in solution. In vivo and in vitro RNA transcriptsof the DNA molecules of the present invention are also encompassed by“isolated” nucleotide sequences. Such polynucleotides are useful in themanufacture of the encoded polypeptide, as probes for isolatinghomologous sequences (e.g., from other mammalian species), for genemapping (e.g., by in situ hybridization with chromosomes), or fordetecting expression of the gene in tissue (e.g., human tissue), such asby Northern blot analysis.

The nucleic acid molecules of the invention can comprise one or moremodified nucleotide residues. The modification may be at the base, sugarand/or phosphate moiety and include, for example, halogenation,hydroxylation, alkylation, an attached linker and/or label. Themodifications can further comprise, for example, labeling, methylation,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates), chargedlinkages (e.g., phosphorothioates, phosphorodithioates), pendentmoieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids). Also included are synthetic molecules that mimic nucleicacid molecules in the ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such moleculesinclude, for example, those in which peptide linkages substitute forphosphate linkages in the backbone of the molecule.

In certain embodiments, nucleic acid molecules of the invention include,but are not limited to, IL4R, IL4 and/or IL13 mRNA, cDNA and/or genomicDNA molecules. The nucleotide sequence of the coding region of a IL4RmRNA is available from GenBank under accession number X52425.1,nucleotides 176-2653 are provided as SEQ ID NO: 2, shown in a 5′ to 3′orientation in Table 1, below. The IL4R mRNA is provided as SEQ IDNO: 1. Although only one strand of the nucleic acid is shown in Table 1,those of skill in the art will recognize that SEQ ID NO: 1 and SEQ IDNO: 2 identify regions of double-stranded genomic nucleic acid, and thatthe sequences of both strands are fully specified by the sequenceinformation provided. The genomic sequence for the IL4R gene is includedin GenBank accession number AC004525.1 (SEQ ID NO: 88). The nucleotidesequence of the coding region of a IL4 mRNA is available from GenBankunder accession number M23442.1 (SEQ ID NO: 89) and the nucleotidesequence of the coding region of a IL13 mRNA is available from GenBankunder accession number U10307.1 (SEQ ID NO: 90).

Primers And Probes

By “oligonucleotide” is meant a single-stranded nucleotide polymer madeof more than 2 nucleotide subunits covalently joined together. In oneembodiment said oligonucleotides are between about 10 and 1000nucleotide units, in a further embodiment, said oligonucleotides arebetween about 12 and 100 nucleotides units. The sugar groups of thenucleotide subunits may be ribose, deoxyribose or modified derivativesthereof such as o-methyl ribose. The nucleotide subunits of anoligonucleotide may be joined by phosphodiester linkages,phosphorothioate linkages, methyl phosphonate linkages or by otherlinkages, including but not limited to rare or non-naturally-occurringlinkages, that do not prevent hybridization of the oligonucleotide.Furthermore, an oligonucleotide may have uncommon nucleotides ornon-nucleotide moieties. An oligonucleotide as defined herein is anucleic acid, preferably DNA, but may be RNA or have a combination ofribo- and deoxyribonucleotides covalently linked. Oligonucleotide probesand amplification oligonucleotides of a defined sequence may be producedby techniques known to those of ordinary skill in the art, such as bychemical or biochemical synthesis, and by in vitro or in vivo expressionfrom recombinant nucleic acid molecules, e.g., bacterial or retroviralvectors. As used herein, an oligonucleotide does not consist ofwild-type chromosomal DNA or the in vivo transcription products thereof.

Primer and probe sequences may comprise DNA, RNA (oligonucleotides—seeabove) or nucleic acid analogs such as uncharged nucleic acid analogsincluding but not limited to peptide nucleic acids (PNAs) which aredisclosed in International Patent Application WO 92/20702 or morpholinoanalogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506, and5,142,047 all of which are herein incorporated by reference in theirentireties. Such sequences can routinely be synthesized using a varietyof techniques currently available. For example, a sequence of DNA can besynthesized using conventional nucleotide phosphoramidite chemistry andthe instruments available from Applied Biosystems, Inc, (Foster City,Calif.); DuPont, (Wilmington, Del.); or Milligen, (Bedford, Mass.).Similarly, and when desirable, the sequences can be labeled usingmethodologies well known in the art such as described in U.S. Pat. Nos.5,464,746; 5,424,414; and 4,948,882 all of which are herein incorporatedby reference in their entireties. Primers and Probes may be used in avariety of ways and may be defined by the specific use. For example, a“capture probe” is immobilized or can be immobilized on a solid supportby any appropriate means, including, but not limited to: by covalentbonding, by adsorption, by hydrophobic and/or electrostatic interaction,or by direct synthesis on a solid support (see in particular patentapplication WO 92 10092). A “detection probe” may be labeled by means ofa marker chosen, for example, from radioactive isotopes, enzymes, inparticular enzymes capable of acting on a chromogenic, fluorigenic orluminescent substrate (in particular a peroxidase or an alkalinephosphatase), chromophoric chemical compounds, chromogenic, fluorigenicor luminescent compounds, analogues of nucleotide bases, and ligandssuch as biotin. A “primer” is a probe comprising, for example, from 10to 100 nucleotide units and having a hybridization specificity underdetermined conditions for the initiation of an enzymatic polymerization,for example in an amplification technique such as PCR (Polymerase ChainReaction), in a process of sequencing, in a method of reversetranscription and the like. One use of a probe is as a hybridizationassay probe; probes may also be used as in vivo or in vitro therapeuticamplification oligomers or antisense agents to block or inhibit genetranscription, or translation in diseased, infected, or pathogeniccells.

All of the oligonucleotides, primers and probes of the presentinvention, whether hybridization assay probes, amplificationoligonucleotides, or helper oligonucleotides, may be modified withchemical groups to enhance their performance or to facilitate thecharacterization of amplification products. For example,backbone-modified oligonucleotides such as those having phosphorothioateor methylphosphonate groups which render the oligonucleotides resistantto the nucleolytic activity of certain polymerases or to nucleaseenzymes may allow the use of such enzymes in an amplification or otherreaction. Another example of modification involves using non-nucleotidelinkers (e.g., Arnold, et al., “Non-Nucleotide Linking Reagents forNucleotide Probes”, EP 0 313 219 hereby incorporated by reference hereinin its entirety) incorporated between nucleotides in the nucleic acidchain which do not interfere with hybridization or the elongation of theprimer. Amplification oligonucleotides may also contain mixtures of thedesired modified and natural nucleotides.

The 3′ end of an amplification oligonucleotide may be blocked to preventinitiation of DNA synthesis as described by McDonough, et al., entitled“Nucleic Acid Sequence Amplification”, WO94/03472 which enjoys commonownership with the present invention and is hereby incorporated byreference herein in its entirety. A mixture of different 3′ blockedamplification oligonucleotides, or of 3′ blocked and unblockedoligonucleotides may increase the efficiency of nucleic acidamplification, as described therein.

The 5′ end of the oligonucleotides may be modified to be resistant tothe 5′-exonuclease activity present in some nucleic acid polymerases.Such modifications can be carried out by adding a non-nucleotide groupto the terminal 5′ nucleotide of the primer using techniques such asthose described by Arnold, et al., supra, entitled “Non-NucleotideLinking Reagents for Nucleotide Probes”, incorporated by referenceherein.

Once synthesized, selected oligonucleotide probes may be labeled by anyof several well-known methods (e.g., J. Sambrook, supra). Useful labelsinclude radioisotopes as well as non-radioactive reporting groups.Isotopic labels include ³H, ³⁵S, ³²P, ¹²⁵I, ⁵⁷Co and ¹⁴C. Isotopiclabels can be introduced into the oligonucleotide by techniques known inthe art such as nick translation, end labeling, second strand synthesis,the use of reverse transcription, and by chemical methods. When usingradiolabeled probes hybridization can be detected by autoradiography,scintillation counting, or gamma counting. The detection method selectedwill depend upon the particular radioisotope used for labeling.

Non-isotopic materials can also be used for labeling and may beintroduced internally into the nucleic acid sequence or at the end ofthe nucleic acid sequence. Modified nucleotides may be incorporatedenzymatically or chemically. Chemical modifications of the probe may beperformed during or after synthesis of the probe, for example, throughthe use of non-nucleotide linker groups as described by Arnold, et al.,supra “Non-Nucleotide Linking Reagents for Nucleotide Probes,”incorporated by reference herein. Non-isotopic labels includefluorescent molecules, chemiluminescent molecules, enzymes, cofactors,enzyme substrates, haptens or other ligands.

In one embodiment, the probes are labeled with an acridinium ester.Acridinium ester labeling may be performed as described by Arnold etal., U.S. Pat. No. 5,185,439, entitled “Acridinium Ester Labeling andPurification of Nucleotide Probes,” issued Feb. 9, 1993 and herebyincorporated by reference herein in its entirety. TABLE 1 SEQ ID NO: 2   1 atggggtggc tttgctctgg gctcctgttc cctgtgagct gcctggtcct gctgcaggtg  61 gcaagctctg ggaacatgaa ggtcttgcag gagcccacct gcgtctccga ctacatgagc 121 atctctactt gcgagtggaa gatgaatggt cccaccaatt gcagcaccga gctccgcctg 181 ttgtaccagc tggtttttct gctctccgaa gcccacacgt gtatccctga gaacaacgga 241 ggcgcggggt gcgtgtgcca cctgctcatg gatgacgtgg tcagtgcgga taactataca 301 ctggacctgt gggctgggca gcagctgctg tggaagggct ccttcaagcc cagcgagcat 361 gtgaaaccca gggccccagg aaacctgaca gttcacacca atgtctccga cactctgctg 421 ctgacctgga gcaacccgta tccccctgac aattacctgt ataatcatct cacCtatgca 481 gtcaacattt ggagtgaaaa cgacccggca gatttcagaa tctataacgt gacctaccta 541 gaaccctccc tccgcatcgc agccagcacc ctgaagtctg ggatttccta cagggcacgg 601 gtgagggcct gggctcagtg ctataacacc acctggagtg agtggagccc cagcaccaag 661 tggcacaact cctacaggga gcccttcgag cagcacctcc tgctgggcgt cagcgtttcc 721 tgcattgtca tcctggccgt ctgcctgttg tgctatgtca gcatcaccaa gattaagaaa 781 gaatggtggg atcagattcc caacccagcc cgcagccgcc tcgtggctat aataatccag 841 gatgctcagg ggtcacagtg ggagaagcgg tcccgaggcc aggaaccagc caagtgccca 901 cactggaaga attgtcttac caagctcttg ccctgttttc tggagcacaa catgaaaagg 961 gatgaagatc ctcacaaggc tgccaaagag atgcctttcc agggctctgg aaaatcagca1021 tggtgcccag tggagatcag caagacagtc ctctggccag agagcatcag cgtggtgcga1081 tgtgtggagt tgtttgaggc cccggtggag tgtgaggagg aggaggaggt agaggaagaa1141 aaagggagct tctgtgcatc gcctgagagc agcagggatg acttccagga gggaagggag1201 ggcattgtgg cccggctaac agagagcctg ttcctggacc tgctcggaga ggagaatggg1261 ggcttttgcc agcaggacat gggggagtca tgccttcttc caccttcggg aagtacgagt1321 gctcacatgc cctgggatga gttcccaagt gcagggccca aggaggcacc tccctggggc1381 aaggagcagc ctctccacct ggagccaagt cctcctgcca gcccgaccca gagtccagac1441 aacctgactt gcacagagac gcccctcgtc atcgcaggca accctgctta ccgcagcttc1501 agcaactccc tgagccagtc accgtgtccc agagagctgg gtccagaccc actgctggcc1561 agacacctgg aggaagtaga acccgagatg ccctgtgtcc cccagctctc tgagccaacc1621 actgtgcccc aacctgagcc agaaacctgg gagcagatcc tccgccgaaa tgtcctccag1681 catggggcag ctgcagcccc cgtctcggcc cccaccagtg gctatcagga gtttgtacat1741 gcggtggagc agggtggcac ccaggccagt gcggtggtgg gcttgggtcc cccaggagag1801 gctggttaca aggccttctc aagcctgctt gccagcagtg ctgtgtcccc agagaaatgt1861 gggtttgggg ctagcagtgg ggaagagggg tataagcctt tccaagacct cattcctggc1921 tgccctgggg accctgcccc agtccctgtc cccttgttca cctttggact ggacagggag1981 ccacctcgca gtccgcagag ctcacatctc ccaagcagct ccccagagca cctgggtctg2041 gagccggggg aaaaggtaga ggacatgcca aagcccccac ttccccagga gcaggccaca2101 gacccccttg tggacagcct gggcagtggc attgtctact cagcccttac ctgccacctg2161 tgcggccacc tgaaacagtg tcatggccag gaggatggtg gccagacccc tgtcatggcc2221 agtccttgct gtggctgctg ctgtggagac aggtcctcgc cccctacaac ccccctgagg2281 gccccagacc cctctccagg tggggttcca ctggaggcca gtctgtgtcc ggcctccctg2341 gcaccctcgg gcatctcaga gaagagtaaa tcctcatcat ccttccatcc tgcccctggc2401 aatgctcaga gctcaagcca gacccccaaa atcgtgaact ttgtctccgt gggacccaca2461 tacatgaggg tctcttag

SNPs

In one aspect, the present invention provides a method for detecting anindividual's increased or decreased risk for an autoimmune disease suchas type 1 diabetes by detecting the presence of one or more IL4R, IL4 orIL13 SNPs in a nucleic acid sample of the individual, wherein thepresence of said SNP(s) indicates the individual's increased ordecreased risk for type 1 diabetes. The SNPs can be any SNPs in theIL4R, IL4 or IL13 loci including SNPs in exons, introns or upstream ordownstream regions. Examples of such SNPs include, but are not limitedto those provided in Table 2, below, and discussed in detail in theExamples. In one embodiment, the SNPs present in the IL4R, IL4 or IL13loci are identified by genotyping the IL4R, IL4 or IL13 SNPs.

In certain embodiments, the genotype of one IL4R, IL4 or IL13 SNP can beused to determine an individual's risk for an autoimmune disease. Inother embodiments, the genotypes of a plurality of IL4R, IL4 or IL13SNPs can be used. For example, in certain embodiments, the genotypes of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 of the SNPs in Table 2 canbe used to determine a individual's risk for an autoimmune disease. Inother embodiments, certain combinations of SNPs at either the same ordifferent loci can be used, as described in the Examples, below. TABLE 2IL4R, IL4 and IL13 SNPs IL4R SNPs Acc dbSNP WT Var X52425.1 AC004525.1Formal ID rs# Exon* Variation allele allele (cDNA) (genomic) SNP name PC(−3223)T G A NA 128387 G128387A P T(−1914)C A G NA 127078 A127078G 3I50V A G  398 94272 A398G 4 N142N C T  676 92548 C676T 4 C92516T C T NA92516 C92516T 4 A92417T A T NA 92417 A92417T 2234896 7 P249P C G  99780189 C997G 2234897 9 F288F T C 1114 76868 T1114C 1805011 9 E375A A C1374 76608 A1374C 9 E375E G A 1375 76607 G1375A 2234898 9 L389L G T 141776565 G1417T 1805012 9 C406R T C 1466 76516 T1466C 2234899 9 C406C C T1468 76514 C1468T 2234900 9 L408L T C 1474 76508 T1474C 1805013 9 S411LC T 1482 76500 C1482T 1805015 9 S478P T C 1682 76300 T1682C 1801275 9Q551R A G 1902 76080 A1902G 9 V554I G A 1910 76072 G1910A 9 P650S C T2198 75784 C2198T 1805016 9 S727A T G 2429 75553 T2429G 9 G759G C T 256775455 C2567T 1805014 9 S761P T C 2531 75451 T2531C 9 P774P T C 257275410 T2572C 1049631 9 3′UTR G A 3044 74938 G3044A   8832 9 3′UTR A G3289 74693 A3289G   8674 9 3′UTR C T 3391 74581 C3391T SNP WT VarGenbank Chromosomal Formal Description Exon Variation allele alleleAccess # Position SNP name IL4 SNPs 5′ (−524) P (−524) C T M23442.1 5q31C582T IL13 SNPs (−1512) P (−1512) A C U10307.1 5q31 A(−1512)C (−1112) P(−1112) C T U10307.1 5q31 C(−1112)T Intron 3 — Intron 3 C T U10307.15q31 C4045T R110Q 4 R110Q G A U10307.1 5q31 G4166AP: Promoter region*The exon numbering is based on the scheme disclosed by Ober etal.,2000, Am J Hum Genet 66: 517-526. Other numbering schemes are availableand one of skill in the art will be able to identify the SNP and thescheme is used.

Genotyping Methods

In the methods of the present invention, the alleles present in a sampleare identified by identifying the nucleotide present at one or more ofthe polymorphic sites. Any type of tissue containing IL4R, IL4 or IL13nucleic acid may be used for determining the IL4R, IL4 or IL13 genotypesof an individual. A number of methods are known in the art foridentifying the nucleotide present at polymorphic sites. The particularmethod used to identify the genotype is not a critical aspect of theinvention. Although considerations of performance, cost, and conveniencewill make particular methods more desirable than others, it will beclear that any method that can identify the nucleotide present willprovide the information needed to identify the genotype. Preferredgenotyping methods involve DNA sequencing, allele-specificamplification, or probe-based detection of amplified nucleic acid.

IL4R, IL4 or IL13 alleles can be identified by DNA sequencing methods,such as the chain termination method (Sanger et al., 1977, Proc. Natl.Acad. Sci,. 74:5463-5467, incorporated herein by reference), which arewell known in the art. In one embodiment, a subsequence of the geneencompassing the polymorphic site is amplified and either cloned into asuitable plasmid and then sequenced, or sequenced directly. PCR-basedsequencing is described in U.S. Pat. No. 5,075,216; Brow, in PCRProtocols, 1990, (Innis et al., eds., Academic Press, San Diego),chapter 24; and Gyllensten, in PCR Technology, 1989 (Erlich, ed.,Stockton Press, New York), chapter 5; each incorporated herein byreference. Typically, sequencing is carried out using one of theautomated DNA sequencers which are commercially available from, forexample, PE Biosystems (Foster City, Calif.), Pharmacia (Piscataway,N.J.), Genomyx Corp. (Foster City, Calif.), LI-COR Biotech (Lincloln,Neb.), GeneSys technologies (Sauk City, Wis.), and Visible Genetics,Inc. (Toronto, Canada).

IL4R, IL4 or IL13 alleles can also be identified usingamplification-based genotyping methods. Various nucleic acidamplification methods known in the art can be used in to detectnucleotide changes in a target nucleic acid. A preferred method is thepolymerase chain reaction (PCR), which is now well known in the art, anddescribed in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; eachincorporated herein by reference. Examples of the numerous articlespublished describing methods and applications of PCR are found in PCRApplications, 1999, (Innis et al., eds., Academic Press, San Diego), PCRStrategies, 1995, (Innis et al., eds., Academic Press, San Diego); PCRProtocols, 1990, (Innis et al., eds., Academic Press, San Diego); andPCR Technology, 1989, (Erlich, ed., Stockton Press, New York); eachincorporated herein by reference. Commercial vendors, such as PEBiosystems (Foster City, Calif.) market PCR reagents and publish PCRprotocols.

Other suitable amplification methods include the ligase chain reaction(Wu and Wallace, 1988, Genomics 4:560-569); the strand displacementassay (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396,Walker et al. 1992, Nucleic Acids Res. 20:1691-1696, and U.S. Pat. No.5,455,166); and several transcription-based amplification systems,including the methods described in U.S. Pat. Nos. 5,437,990; 5,409,818;and 5,399,491; the transcription amplification system (TAS) (Kwoh etal., 1989, Proc. Natl. Acad. Sci. USA, 86:1173-1177); and self-sustainedsequence replication (3SR) (Guatelli et al., 1990, Proc. Natl. Acad.Sci. USA, 87:1874-1878 and WO 92/08800); each incorporated herein byreference. Alternatively, methods that amplify the probe to detectablelevels can be used, such as QB-replicase amplification (Kramer et al.,1989, Nature, 339:401-402, and Lomeli et al., 1989, Clin. Chem.,35:1826-1831, both of which are incorporated herein by reference). Areview of known amplification methods is provided in Abramson et al.,1993, Current Opinion in Biotechnology, 4:41-47, incorporated herein byreference.

Genotyping also can also be carried out by detecting and analyzing IL4R,IL4 or IL13 mRNA under conditions when both, maternal and paternal,chromosomes are transcribed. Amplification of RNA can be carried out byfirst reverse-transcribing the target RNA using, for example, a viralreverse transcriptase, and then amplifying the resulting cDNA, or usinga combined high-temperature reverse-transcription-polymerase chainreaction (RT-PCR), as described in U.S. Pat. Nos. 5,310,652; 5,322,770;5,561,058; 5,641,864; and 5,693,517; each incorporated herein byreference (see also Myers and Sigua, 1995, in PCR Strategies, supra,chapter 5).

IL4R, IL4 or IL13 alleles can also be identified using allele-specificamplification or primer extension methods, which are based on theinhibitory effect of a terminal primer mismatch on the ability of a DNApolymerase to extend the primer. To detect an allele sequence using anallele-specific amplification or extension-based method, a primercomplementary to the IL4R, IL4 or IL13 genes is chosen such that the 3′terminal nucleotide hybridizes at the polymorphic position. In thepresence of the allele to be identified, the primer matches the targetsequence at the 3′ terminus and primer is extended. In the presence ofonly the other allele, the primer has a 3′ mismatch relative to thetarget sequence and primer extension is either eliminated orsignificantly reduced. Allele-specific amplification- or extension-basedmethods are described in, for example, U.S. Pat. Nos. 5,137,806;5,595,890; 5,639,611; and U.S. Pat. No. 4,851,331, each incorporatedherein by reference.

Using allele-specific amplification-based genotyping, identification ofthe alleles requires only detection of the presence or absence ofamplified target sequences. Methods for the detection of amplifiedtarget sequences are well known in the art. For example, gelelectrophoresis (see Sambrook et al., 1989, supra.) and the probehybridization assays described above have been used widely to detect thepresence of nucleic acids.

Allele-specific amplification-based methods of genotyping can facilitatethe identification of haplotypes, as described in the examples.Essentially, the allele-specific amplification is used to amplify aregion encompassing multiple polymorphic sites from only one of the twoalleles in a heterozygous sample. The SNP variants present within theamplified sequence are then identified, such as by probe hybridizationor sequencing.

An alternative probe-less method, referred to herein as a kinetic-PCRmethod, in which the generation of amplified nucleic acid is detected bymonitoring the increase in the total amount of double-stranded DNA inthe reaction mixture, is described in Higuchi et al., 1992,Bio/Technology, 10:413-417; Higuchi et al., 1993, Bio/Technology,11:1026-1030; Higuchi and Watson, in PCR Applications, supra, Chapter16; U.S. Pat. Nos. 5,994,056 and 6,171,785; and European PatentPublication Nos. 487,218 and 512,334, each incorporated herein byreference. The detection of double-stranded target DNA relies on theincreased fluorescence that DNA-binding dyes, such as ethidium bromide,exhibit when bound to double-stranded DNA. The increase ofdouble-stranded DNA resulting from the synthesis of target sequencesresults in an increase in the amount of dye bound to double-stranded DNAand a concomitant detectable increase in fluorescence. For genotypingusing the kinetic-PCR methods, amplification reactions are carried outusing a pair of primers specific for one of the alleles, such that eachamplification can indicate the presence of a particular allele. Bycarrying out two amplifications, one using primers specific for thewild-type allele and one using primers specific for the mutant allele,the genotype of the sample with respect to that SNP can be determined.Similarly, by carrying out four amplifications, each with one of thepossible pairs possible using allele specific primers for both theupstream and downstream primers, the genotype of the sample with respectto two SNPs can be determined. This gives haplotype information for apair of SNPs.

Alleles can be also identified using probe-based methods, which rely onthe difference in stability of hybridization duplexes formed between aprobe and its corresponding target sequence comprising an IL4R, IL4 orIL13 allele. Under sufficiently stringent hybridization conditions,stable duplexes are formed only between a probe and its target allelesequence and not other allele sequences. The presence of stablehybridization duplexes can be detected by any of a number of well knownmethods. In general, it is preferable to amplify a nucleic acidencompassing a polymorphic site of interest prior to hybridization inorder to facilitate detection. However, this is not necessary ifsufficient nucleic acid can be obtained without amplification.

A probe suitable for use in the probe-based methods of the presentinvention, which contains a hybridizing region either substantiallycomplementary or exactly complementary to a target region of SEQ ID NOS:2, 88, 89 or 90 or the complement of SEQ ID NOS: 2, 88, 89 or 90,wherein the target region encompasses the polymorphic site, and exactlycomplementary to one of the two allele sequences at the polymorphicsite, can be selected using the guidance provided herein and well knownin the art. Similarly, suitable hybridization conditions, which dependon the exact size and sequence of the probe, can be selected empiricallyusing the guidance provided herein and well known in the art. The use ofoligonucleotide probes to detect nucleotide variations including singlebase pair differences in sequence is described in, for example, Conneret al., 1983, Proc. Natl. Acad. Sci. USA, 80:278-282, and U.S. Pat. Nos.5,468,613 and 5,604,099, each incorporated herein by reference.

In preferred embodiments of the probe-based methods for determining theIL4R, IL4 or IL13 genotypes, multiple nucleic acid sequences from theIL4R, IL4 or IL13 genes which encompass the polymorphic sites areamplified and hybridized to a set of probes under sufficiently stringenthybridization conditions. The alleles present are inferred from thepattern of binding of the probes to the amplified target sequences. Inthis embodiment, amplification is carried out in order to providesufficient nucleic acid for analysis by probe hybridization. Thus,primers are designed such that regions of the IL4R, IL4 or IL13 genesencompassing the polymorphic sites are amplified regardless of theallele present in the sample. Allele-independent amplification isachieved using primers which hybridize to conserved regions of the IL4R,IL4 or IL13 genes. The IL4R, IL4 or IL13 genes contain many invariant ormonomorphic regions and suitable allele-independent primers can beselected routinely from SEQ ID NOS: 1, 88, 89 or 90. One of skill willrecognize that, typically, experimental optimization of an amplificationsystem is helpful.

Suitable assay formats for detecting hybrids formed between probes andtarget nucleic acid sequences in a sample are known in the art andinclude the immobilized target (dot-blot) format and immobilized probe(reverse dot-blot or line-blot) assay formats. Dot blot and reverse dotblot assay formats are described in U.S. Pat. Nos. 5,310,893; 5,451,512;5,468,613; and 5,604,099; each incorporated herein by reference.

In a dot-blot format, amplified target DNA is immobilized on a solidsupport, such as a nylon membrane. The membrane-target complex isincubated with labeled probe under suitable hybridization conditions,unhybridized probe is removed by washing under suitably stringentconditions, and the membrane is monitored for the presence of boundprobe. A preferred dot-blot detection assay is described in theexamples.

In the reverse dot-blot (or line-blot) format, the probes areimmobilized on a solid support, such as a nylon membrane or a microtiterplate. The target DNA is labeled, typically during amplification by theincorporation of labeled primers. One or both of the primers can belabeled. The membrane-probe complex is incubated with the labeledamplified target DNA under suitable hybridization conditions,unhybridized target DNA is removed by washing under suitably stringentconditions, and the membrane is monitored for the presence of boundtarget DNA. A preferred reverse line-blot detection assay is describedin the examples.

Probe-based genotyping can be carried out using a “TaqMan” or“5′-nuclease assay,” as described in U.S. Pat. Nos. 5,210,015;5,487,972; and 5,804,375; and Holland et al., 1988, Proc. Natl. Acad.Sci. USA, 88:7276-7280, each incorporated herein by reference. In theTaqMan assay, labeled detection probes that hybridize within theamplified region are added during the amplification reaction mixture.The probes are modified so as to prevent the probes from acting asprimers for DNA synthesis. The amplification is carried out using a DNApolymerase that possesses 5′ to 3′ exonuclease activity, e.g., Tth DNApolymerase. During each synthesis step of the amplification, any probewhich hybridizes to the target nucleic acid downstream from the primerbeing extended is degraded by the 5′ to 3′ exonuclease activity of theDNA polymerase. Thus, the synthesis of a new target strand also resultsin the degradation of a probe, and the accumulation of degradationproduct provides a measure of the synthesis of target sequences.

Any method suitable for detecting degradation product can be used in theTaqMan assay. In a preferred method, the detection probes are labeledwith two fluorescent dyes, one of which is capable of quenching thefluorescence of the other dye. The dyes are attached to the probe,preferably one attached to the 5′ terminus and the other is attached toan internal site, such that quenching occurs when the probe is in anunhybridized state and such that cleavage of the probe by the 5′ to 3′exonuclease activity of the DNA polymerase occurs in between the twodyes. Amplification results in cleavage of the probe between the dyeswith a concomitant elimination of quenching and an increase in thefluorescence observable from the initially quenched dye. Theaccumulation of degradation product is monitored by measuring theincrease in reaction fluorescence. U.S. Pat. Nos. 5,491,063 and5,571,673, both incorporated herein by reference, describe alternativemethods for detecting the degradation of probe which occurs concomitantwith amplification.

The TaqMan assay can be used with allele-specific amplification primerssuch that the probe is used only to detect the presence of amplifiedproduct. Such an assay is carried out as described for thekinetic-PCR-based methods described above. Alternatively, the TaqManassay can be used with a target-specific probe.

Examples of other techniques that can be used for probe-based genotypinginclude, but are not limited to, Amplifluor™, Dye Binding-Intercalation,Fluorescence Resonance Energy Transfer (FRET), Hybridization SignalAmplification Method (HSAM), HYB Probes™, Invader/Cleavase Technology(Invader/CFLP™), Molecular Beacons™, Origen™, DNA-Based RamificationAmplification (RAM™), Rolling circle amplification (RCA™), Scorpions™,Strand displacement amplification (SDA).

The assay formats described above typically utilize labeledoligonucleotides to facilitate detection of the hybrid duplexes.Oligonucleotides can be labeled by incorporating a label detectable byspectroscopic, photochemical, biochemical, immunochemical, radiological,radiochemical or chemical means. Useful labels include ³²P, fluorescentdyes, electron-dense reagents, enzymes (as commonly used in ELISAs),biotin, or haptens and proteins for which antisera or monoclonalantibodies are available. Labeled oligonucleotides of the invention canbe synthesized and labeled using the techniques described above forsynthesizing oligonucleotides. For example, a dot-blot assay can becarried out using probes labeled with biotin, as described in Levensonet al., 1989, in PCR Protocols: A Guide to Methods and Applications(Innis et al., eds., Academic Press. San Diego), pages 99-112,incorporated herein by reference. Following hybridization of theimmobilized target DNA with the biotinylated probes undersequence-specific conditions, probes which remain bound are detected byfirst binding the biotin to avidin-horseradish peroxidase (A-HRP) orstreptavidin-horseradish peroxidase (SA-HRP), which is then detected bycarrying out a reaction in which the HRP catalyzes a color change of achromogen.

Whatever the method for determining which oligonucleotides of theinvention selectively hybridize to IL4R, IL4 or IL13 allelic sequencesin a sample, the central feature of the typing method involves theidentification of the IL4R, IL4 or IL13 alleles present in the sample bydetecting the variant sequences present.

The present invention also relates to a kit, a container unit comprisinguseful components for practicing the present method. A useful kit cancontain oligonucleotide probes specific for IL4R, IL⁴ or IL13 alleles aswell as instructions for their use to determine risk for an autoimmunedisease such as type 1 diabetes. In some cases, detection probes may befixed to an appropriate support membrane. The kit can also containamplification primers for amplifying regions of the IL4R, IL4 or IL13loci encompassing the polymorphic sites, as such primers are useful inthe preferred embodiment of the invention. Alternatively, useful kitscan contain a set of primers comprising an allele-specific primer forthe specific amplification of IL4R, IL4 or IL13 alleles. Other optionalcomponents of the kits include additional reagents used in thegenotyping methods as described herein. For example, a kit additionallycan contain an agent to catalyze the synthesis of primer extensionproducts, substrate nucleoside triphosphates, reagents for labelingand/or detecting nucleic acid (for example, an avidin-enzyme conjugateand enzyme substrate and chromogen if the label is biotin) andappropriate buffers for amplification or hybridization reactions.

The present invention also relates to an array, a support withimmobilized oligonucleotides useful for practicing the present method. Auseful array can contain oligonucleotide probes specific for IL4R, IL4,IL13 alleles or certain combinations of IL4R, IL4 and/or IL13 alleles.The oligonucleotides can be immobilized on a substrate, e.g., a membraneor glass. The oligonucleotides can, but need not, be labeled. In someembodiments, the array can be a micro-array. In some embodiments, thearray can comprise one or more oligonucleotides used to detect thepresence of two or more IL4R, IL4, IL13 alleles or certain combinationsof IL4R, IL4 and/or IL13 alleles.

The examples of the present invention presented below are provided onlyfor illustrative purposes and not to limit the scope of the invention.Numerous embodiments of the invention within the scope of the claimsthat follow the examples will be apparent to those of ordinary skill inthe art from reading the foregoing text and following examples.

7 EXAMPLE 1 Genotyping Protocol: Probe-Based Identification of IL4R, IL4and IL13 Alleles

This example describes a method of genotyping SNPs in the IL4R, IL4 andIL13 loci that are associated with type 1 diabetes. Two differentgenotyping methods, line blot assays and kinetic thermocycling, wereused, depending on the region and gene genotyped.

Line Blot Assay for Identifying 8 IL4R SNPs, 1 IL4 SNP and 2 IL13 SNPs

Eight exemplary SNPs in the human IL4R gene (listed in Table 6), oneexemplary SNP in the human IL4 gene (Table 2) and two exemplary SNPs inthe human IL4R gene (Table 2) were genotyped using this method. Each SNPis described by its position in the reference GenBank accessionsequence. For example, SNP 1 of Table 6 is found at position 398 ofX52425.1 (SEQ ID NO: 1), where an “A” nucleotide is present. The variantallele at this position has a “G” nucleotide. The SNPs will be referredto by the SNP # in the subsequent text.

The regions of the IL4R, IL4 and IL13 genes that encompass the SNPs wereamplified and the nucleotide present identified by probe hybridization.The probe detection was carried out using an immobilized probe (lineblot) format.

Amplicons and Primers

The pairs of primers used to amplify the regions encompassing the eightIL4R SNPs are listed in Table 7 (SEQ ID NO: 25-36) and those used toamplify the regions encompassing the IL4 SNP and two IL13 SNPs arelisted in Table 30 (SEQ ID NO: 69-74). IL4R SNP numbers 3, 4, and 5(Table 6) were co-amplified on the same 228 basepair fragment. Theprimers were modified at the 5′ phosphate by conjugation with biotin.Reagents for synthesizing oligonucleotides with a biotin label attachedto the 5′ phosphate are commercially available from Clontech (Palo Alto,Calif.) and Glenn Research (Sterling, Va.). A preferred reagent isBiotin-ON from Clontech.

Amplification Primers

Amplification of six regions of the IL4R gene, which encompass eightpolymorphic sites, the one region of the IL4 gene, which encompass onepolymorphic site, and the two regions of the IL13 gene, which encompasstwo polymorphic sites, was carried out using the primer pairs shownbelow. All primers are shown in the 5′ to 3′ orientation.

The following primers amplify a 114 base-pair region encompassingnucleotide position 398 the IL4R gene. RR192B (SEQ ID NO: 25)CAGCCCCTGTGTCTGCAGA RR193B (SEQ ID NO: 31) GTCCAGTGTATAGTTATCCGCACTGA

The following primers amplify a 163 base-pair region encompassingnucleotide position 676. DBM0177B (SEQ ID NO: 26) CTGACCTGGAGCAACCCGTADBM0178B (SEQ ID NO: 32) ACTGGGCCTCTGCTGGTCA

The following primers amplify a 228 base-pair region encompassingnucleotide positions 1374, 1417, and 1466 of the IL4R gene. DBM0023B(SEQ ID NO: 27) ATTGTGTGAGGAGGAGGAGGAGGTA DBM0022B (SEQ ID NO: 33)GTTGGGCATGTGAGCACTCGTA

The following primers amplify a 129 base-pair region encompassingnucleotide position 1682 of the IL4R gene. DBM0097B (SEQ ID NO: 28)CTCGTCATCGCAGGCAA DBM0098B (SEQ ID NO: 34) AGGGCATCTCGGGTTCTA

The following primers amplify a 198 base-pair region encompassingnucleotide position 1902 of the IL4R gene. RR200B (SEQ ID NO: 29)GCCGAAATGTCCTCCAGCA RR178B (SEQ ID NO: 35) CCACATTTCTCTGGGGACACA

The following primers amplify a 177 base-pair region encompassingnucleotide position 2531 of the IL4R gene. DBM0112B (SEQ ID NO: 30)CCGGCCTCCCTGGCA DBM0071B (SEQ ID NO: 36) GCAGACTCAGCAACAAGAGG

The following primers amplify a 107 base-pair region encompassingnucleotide position 582 in the promoter region of the IL4 gene. RR169B(SEQ ID NO: 69) ACTAGGCCTCACCTGATACGA RR170B (SEQ ID NO: 72)CATAGAGGCAGAATAACAGGCAGA

The following primers amplify a 118 base-pair region encompassingnucleotide position 4045 in intron 3 of the IL13 gene. DBM0165B (SEQ IDNO: 70) CTCGGACATGCAAGCTGGAA DBM0166B (SEQ ID NO: 73)ACTGAATGAGACAGTCCCTGGA

The following primers amplify a 187 base-pair region encompassing codon4166 in exon 4 of the IL13 gene. DBM0167B (SEQ ID NO: 71)AATCGAGGTGGCCCAGTTTGTA DBM0168B (SEQ ID NO: 74) CCTAACCCTCCTTCCCGCCTAAmplification

The PCR amplification was carried out in a total reaction volume of25-100 μl containing the following reagents:

0.2 ng/μl purified human genomic DNA

0.2 mM each primer

800 mM total dNTP (200 mM each dATP, dTTP, dCTP, dGTP)

70 mM KCl

12 mM Tris-HCl, pH 8.3

3 mM MgCl₂,

0.25 units/μl AmpliTaq Gold™ DNA polymerase** developed and manufactured by Hoffmann-La Roche and commerciallyavailable from Applera (Foster City, Calif.).

Amplification was carried out in a GeneAmp™ PCR System 9600 thermalcycler (Applera, Foster City, Calif.), using the specific temperaturecycling profile shown below. Pre-reaction incubation: 94° C. for 12.5minutes 33 cycles: denature: 95° C. for 45 seconds anneal: 61° C. for 30seconds extend: 72° C. for 45 seconds Final extension: 72° C. for 7minutes Hold: 10° C.-15° C.Detection Probes

Preferred probes used to identify the nucleotides present at the 8 SNPspresent in the amplified IL4R nucleic acids are described in Table 3.Two probes are shown for the detection of T1466; a mixture of the twoprobes was used. Preferred probes used to identify the nucleotidespresent at the one SNP present in the amplified IL4 nucleic acids andthe two SNPs present in the amplified IL13 nucleic acids are describedin Table 29. All probes are shown in the 5′ to 3′ orientation.

Probe Hybridization Assay, Immobilized Probe Format

In the immobilized probe format, the probes were immobilized to a solidsupport prior to being used in the hybridization. The probe-supportcomplex was immersed in a solution containing denatured amplifiednucleic acid (biotin labeled) to allow hybridization to occur. Unboundnucleic acid was removed by washing under stringent hybridizationconditions, and nucleic acid remaining bound to the immobilized probeswas detected using a chromogenic reaction. The details of the assay aredescribed below.

For use in the immobilized probe detection format, described below, amoiety was attached to the 5′ phosphate of the probe to facilitateimmobilization on a solid support. See Cheng et al., 1999, Genome Res9:936-949, incorporated herein by reference. Preferably, Bovine SerumAlbumin (BSA) is attached to the 5′ phosphate essentially as describedby Tung et al., 1991, Bioconjugate Chem., 2:464-465, incorporated hereinby reference. Alternatively, a poly-T tail is added to the 5′ end asdescribed in U.S. Pat. No 5,451,512, incorporated herein by reference.

The probes were applied in a linear format to sheets of nylon membrane(e.g., BioDyne™ B nylon filters, Pall Corp., Glen Cove, N.Y.) using aLinear Striper and Multispense2000™ controller (IVEK, N. Springfield,Vt.). The detection of the wildtype allele of SNP #5 (table 6) wascarried out using a mixture of two probes as listed; this mixtureenables the detection of SNP #5 indiscriminately of another nearby SNP.Probe titers were chosen to achieve signal balance between the allelicvariants; the titers used are provided in the table of probes, above.Each sheet was cut to strips between 0.35 and 0.5 cm in width. Todenature the amplification products, 20 μl of amplification product(based on a 50 μl reaction) were added to 20 μl of denaturation solution(1.6% NaOH) and incubated at room temperature to complete denaturation.

The denatured amplification product (40 μl) was added to the well of atyping tray containing 3 ml of hybridization buffer (4×SSPE, 0.5% SDS)and the membrane strip. Hybridizations were allowed to proceed for 15minutes at 55° C. in a rotating water bath. Following hybridization, thehybridization solution was aspirated, the strip was rinsed in 3 ml warmwash buffer (2×SSPE, 0.5% SDS) by gently rocking strips back and forth,and the wash buffer was aspirated. Following rinsing, the strips wereincubated in 3 ml enzyme conjugate solution (3.3 ml hybridization bufferand 12 μl of strepavidin-horseradish peroxidase (SA-HRP)) in therotating water bath for 5 minutes at 55° C. Then the strips were rinsedwith wash buffer, as above, incubated in wash buffer at 55° for 12minutes (stringent wash), and finally rinsed with wash buffer again.

Target nucleic acid, now HRP-labeled, which remains bound to theimmobilized amplification product was visualized as follows. A colordevelopment solution was prepared by mixing 100 ml of citrate buffer(0.1 M Sodium Citrate, pH 5.0), 5 ml 3,3′,5,5′-tetramethylbenzidine(TMB) solution (2 mg/ml TMB powder from Fluka, Milwaukee, Wis.,dissolved in 100% EtOH), and 100 μl of 3% hydrogen peroxide. The stripswere first rinsed in 0.1 M sodium citrate (pH 5.0) for 5 minutes, thenincubated in the color development solution with gentle agitation for 8to 10 minutes at room temperature in the dark. The TMB, initiallycolorless, is converted by the target-bound HRP, in the presence ofhydrogen peroxide, into a colored precipitate. The developed strips wererinsed in water for several minutes and immediately photographed.

Kinetic Thermocycling to Identify 2 IL4R Promoter and 2 IL13 PromoterSNPs

The two IL4R promoter and the two IL13 promoter SNPs were genotypedusing allele-specific PCR on a PE9700 thermal cycler (ABI) measuringSyBr Green (Molecular Probes) fluorescence (Higuchi, Fockler, Dollinger,Watson. Biotechnology 11:1026-30 (1993)). For each DNA, twoampifications were set up in parallel. One contained the common primerand one allele-specific primer; the other contained the common primerand the other allele-specific primer. The primers used to genotype thetwo IL4R promoter SNPs are provided in Table 31 and the primers used togenotype the two IL13 promoter SNPs are provided in Table 32. Theamplification of the DNA with a particular allele-specific primerindicated the presence of the corresponding allele. An increase in thefluorescence of SyBr Green was indicative of the accumulation ofamplification product. One of skill in the art will be able to correlatethe change in fluorescence with the presence or absence of amplificationproduct, and thus, the presence or absence of the corresponding allele.

The PCR amplification was carried out in a total reaction volume of 100μl containing the following reagents: Volume (μl) Component  1 1 M TrispH 8.0 12 25 mM MgCl2  1 100X dNTPs (50 mM each dA, dC, dG; 25 mM dT; 75mM dU)  1 20X SYBR Green  1 200 μM ROX  2 1 U/μl UNG  2.5 80% Glycerol 4 100% DMSO  1 20 μM primer 1  1 20 μM primer 2  1 12 U/μl CEA2 GoldDNA Polymerase*  2 10 ng/μl genomic DNA to 100 μl sterile water*Developed and manufactured by Roche Molecular Systems. Alternatively, 1μl of AmpliTaq Gold DNA Polymerase, also developed and manufactured byRoche Molecular Systems and sold commercially by Applied Biosystems,Inc, (Foster City, Calif.), can be used.

Amplification was carried out in a GeneAmp™ PCR System 9600 thermalcycler (Applera, Foster City, Calif.), using the specific temperaturecycling profile shown below.  2 minutes at 50° C. Pre-reactionincubation: 12 minutes at 94° C. 50 cycles: denature: 20 seconds at 95°C. anneal: 20 seconds at 58° C. Final extension:  5 minutes at 72° C.

8 EXAMPLE 2 Association of IL4R SNPs with Type 1 Diabetes in HBDIFamilies

This example demonstrates the association of IL4R SNPs with type 1diabetes in HBDI families.

IL4R genotyping was carried out on individuals from 282 Caucasianfamilies ascertained because they contained two offspring affected withtype 1 diabetes. The IL4R genotypes of all individuals were determined.IL4R genotyping was carried out using a genotyping method essentially asdescribed in Example 1. In addition to the 564 offspring (2 siblings ineach of 282 families) in the affected sibling pairs on whichascertainment was based, there were 26 other affected children. Therewere 270 unaffected offspring among these families.

The family-based samples were provided as purified genomic DNA from theHuman Biological Data Interchange (HBDI), which is a repository for celllines from families affected with type 1 diabetes. All of the HBDIfamilies used in this study are nuclear families with unaffected parents(genetically unrelated) and at least two affected siblings. Thesesamples are described further in Noble et al., 1996, Am. J. Hum. Genet.59:1134-1148, incorporated herein by reference.

It is known that the HLA genotype can have a significant effect, eitherincreased or decreased depending on the genotype, on the risk for type 1diabetes. In particular, individuals with the HLA DR genotypeDR3-DQB1*0201/DR4-DQB1*0302 (referred to as DR3/DR4 below) appear to beat the highest risk for type 1 diabetes (see Noble et al., 1996, Am. J.Hum. Genet., 59:1134-1148, incorporated herein by reference). Thesehigh-risk individuals have about a 1 in 15 chance of being affected withtype 1 diabetes. Because of the strong effect of this genotype on thelikelihood of type 1 diabetes, the presence of theDR3-DQB1*0201/DR4-DQB1*0302 genotype could mask the contribution fromthe IL4R allelic variants.

Individuals within these families also were genotyped at the HLA DRB1and DQB1 loci. Of the affected sibling pairs, both siblings have theDR3/DR4 genotype in 90 families. Neither affected sibling has the DR 3/4genotype in 144 families. Exactly one of the affected pair has the DR3/4 genotype in the remaining 48 families.

Statistical Analysis, Methods and Algorithms

Since the eight SNPs in IL4R are both physically and genetically veryclosely linked to each other, the presence of a particular allele at aparticular SNP is correlated with the presence of another particularallele at a nearby SNP. This non-random association of two or more SNPs'alleles is known as linkage disequilibrium (LD).

Linkage disequilibrium among the eight IL4R SNPs was assessed using thegenotypes of the 282 pairs of parents. These 564 individuals are notrelated to each other except by marriage. A summary of the calculatedfrequency of the WT allele for each SNP in this group of 564 individuals(the “HBDI founders”) is shown in Table 9.

The calculation of LD can be performed in several ways. Twocomplementary methods to assess LD between all pairs of IL4R SNP lociwere used. In the first method, the values of two distinct but relatedmetrics for LD, namely D and Δ (Devlin and Risch 1995, Genomics, 29(2):311-22), using the Maximum Likelihood Estimation algorithm of Hill(Hill, 1974, Heredity, 33(2): 229-39) were calculated. The values for Dand Δ for all pairs of IL4R SNPs are shown in Table 10, in the lowerleft triangular portion. Both D and Δ can have values that range between−1 and +1. Values near +1 or −1 suggest strong linkage disequilibrium;values near zero indicate the absence of LD.

A second measure of LD uses a permutation test method implemented in theArlequin program (Excoffier et al., 1995, Mol Biol Evol, 12:921-7,University of Geneva, CH) (Slatkin et al., 1996, Heredity, 76:377-83).This method maximizes the likelihood ratio statistic (S=−2log(L_(H*)/L_(H))) by permuting alleles and recalculating S over a largenumber of iterations until S is maximized. These iterations allow thedetermination of the null distribution of S, and thus the maximum Sobtained can be converted into an exact P-value (significance level).These P-values are listed in the upper right triangular portion of Table10.

Table 10 of pairwise LD shows that there is significant evidence for LDbetween SNPs 1 and 2, and among (all combinations of) SNPs 3, 4, 5, 6, 7and 8. SNPs 3 through 8 are known to exist within 1200 basepairs of eachother in a single exon (exon 9) of the IL4R gene, and the LD betweenthese SNPs is evidence for very small genetic distances as well.

The Transmission Disequilibrium Test (TDT) of Spielman (Spielman andEwens, 1996, Am J Hum Genet, 59(5): 983-9; Spielman and Ewens, 1998, AmJ Hum Genet, 62(2): 450-8) was performed on the IL4R genotype data forthe 282 affected sib pairs (namely, a family structure consisting of thetwo parents and the two affected children). The TDT was used to test forthe association of the individual alleles of the eight IL4R SNPs to type1 diabetes. The TDT assesses whether an allele is transmitted fromheterozygous parents to their affected children at a frequency that issignificantly different than expected by chance. Under the nullhypothesis of no association of an allele with disease, a heterozygousparent will transmit or will not transmit an allele with equal frequencyto an affected child. The significance of deviation from the nullhypothesis can be assessed using the McNemar chi-squared test statistic(=(T−NT)ˆ2/(T+NT), where T is the observed number of transmissions andNT is the observed number of non-transmissions). The significance(P-value) of the McNemar chi-squared test statistic is equal to thePearson chi-squared statistic with one degree of freedom (Glantz et al.,Primer of biostatistics., New York, McGraw-Hill Health ProfessionsDivision, 1997).

The results of the single SNP locus TDT results are shown in tables 11Aand 11B. The TDT/S-TDT program (version 1.1) of Spielman was used toperform the counting of transmitted and non-transmitted alleles(Spielman, McGinnis et al., 1993, Am J Hum Genet, 52(3): 506-16;Spielman and Ewens, 1998, supra). The table lists the observedtransmissions of the wildtype allele at each SNP locus. Since these arebiallelic polymorphisms, the transmission counts of the variant alleleare equal to the non-transmissions of the wildtype allele.

The counts of transmissions and non-transmissions of alleles to theprobands only shown in Table 11A do not quite reach statisticalsignificance, at a=0.05. However, it is valid to count transmissionevents to all affected children. However, when the TDT is used in thisway (or, for that matter, with more than one child per family), then asignificant test statistic is evidence of linkage only, not ofassociation and linkage. Table 11B shows the TDT analysis when 26additional affected children are included. The results presented inTable 11B below show that there is a significant deviation from theexpected transmission frequencies for alleles of SNPs 3, 4, 5 and 6.Inspection of the “% transmission” values for these SNPs indicates thatthe wildtype allele is transmitted to affected children at frequenciesgreater than the expectation of 50%.

The evidence for strong LD among the eight IL4R SNPs suggested that thetransmission of the ordered set of alleles from each parent to eachaffected child in the HBDI cohort could be detected. This ordered set ofalleles corresponds physically to one of the two parental chromosomes,and is called a haplotype. By inferring the parental haplotypes andtheir transmission or non-transmission to affected children, morestatistical information is expected to be obtained than that fromalleles alone.

Haplotypes were inferred using a combination of two methods. As thefirst step, the GeneHunter program (Falling Rain Genomics, Palo Alto,Calif.) (Kruglyak, et al., 1996, Am J Hum Genet, 58(6): 1347-63) wasused as it very rapidly calculates haplotypes from genotype data frompedigrees. Each HBDI family pedigree was then inspected individuallyusing the Cyrillic program (Cherwell Scientific Publishing, Palo Alto,Calif.), to resolve any ambiguous or unsupported haplotype assignments.Unambiguous and non-recombinant haplotypes could be confidently assignedin all but six of the 282 families. The haplotype data for these 276families were used in subsequent data analysis.

The IL4R gene has the property that many of the SNPs reside within the3′-most exon (exon 9), whose coding region is approximately 1.5 kb long.A method was developed for directly haplotyping up to five of these exon9 alleles (namely, SNPs #3-7) without needing parental genotypes. Asmany of these SNPs direct changes to the amino acid sequence of the IL4Rprotein, different haplotypes encode different proteins with likelydifferent functions.

Haplotypes, in an individual for which no parental genotypic informationis known, can be inferred unambiguously only when at most one of the SNPsites of those is heterozygous. In other cases, the ambiguity must beresolved experimentally.

Two allele-specific primers with one common primer to perform PCRreactions (using Stoffel Gold™ polymerase) to separately amplify the DNAfrom each chromosome, as shown in FIG. 1 below were used. The alleles oneach amplicon were then detected by the same strip hybridizationprocedure, and the linked alleles called directly. The choice ofallele-specific (colored or shaded arrows) and common (black arrows)primers depends on which SNP loci are heterozygous. The primers weremodified at the 5′ phosphate by conjugation with biotin, and are shownin Table 12 (SEQ ID NO: 54-62).

For each haplotyping assay, two PCR reactions were set up for each DNAto be tested. One reaction contained the common primer and the wildtypeallele-specific primer, the other contained the common primer and thevariant allele-specific primer. Each PCR reaction was made in a totalreaction volume of 50-100 μl containing the following reagents:

-   0.2 ng/ml purified human genomic DNA-   0.2 mM each primer-   800 mM total dNTP (200 mM each dATP, dTTP, dCTP, dGTP)-   10 mM KCl-   10 mM Tris-HCl, pH 8.0-   2.5 mM MgCl₂-   0.12 units/ml Stoffel Gold™ DNA polymerase*    *developed and manufactured by Roche Molecular Systems.

Amplification was carried out in a GeneAmp™ PCR System 9600 thermalcycler (PE Biosystems, Foster City, Calif.), using the specifictemperature cycling profile shown below: Pre-reaction incubation: 94° C.for 12.5 minutes 33 cycles: Denature: 95° C., 45 seconds Anneal: 64° C.,30 seconds Extend: 72° C., 45 seconds Final Extension: 72° C., 7minutes. Hold: 10° C.-15° C.

Following amplification, each PCR product reaction was denatured andseparately used for hybridization to the membrane-bound probes asdescribed above.

Haplotype Sharing in Affected Sibs

Evidence for linkage of IL4R to type 1 diabetes (as opposed toassociation) can be assessed by the haplotype sharing method. Thismethod assesses the distribution over all families of the number ofchromosomes that are identical-by-descent (IBD) between the two affectedsiblings in each family. For example, if in a family, the fathertransmits the same one of his two IL4R haplotypes to both children, andthe mother transmits the same one of her two IL4R haplotypes to bothchildren, then the children are said to share two chromosomes IBD (or,to be IBD=2). If both parents transmit different IL4R haplotypes totheir two children, the children are said to be IBD=0.

Under the null hypothesis of no linkage of IL4R to type 1 diabetes, theproportion of families IBD=0 is 25%, IBD=1 is 50% and IBD=2 is 25%, asexpected by random assortment (see Table 13). Evidence for astatistically significant difference from this expectation can beassessed using the chi-square statistic.

Identity-by-descent (IBD) values of parental IL4R haplotypes in theaffected sibs could be determined unambiguously in 256 families. In therest of the families, one or both parents were homozygous and/or theparental source of the child's chromosomes could not be determined. Thedistribution of IBD is shown in Table 13.

It is known that the HLA genotype can have a significant effect, eitherincreased or decreased depending on the genotype, on the risk for type 1diabetes. In particular, individuals with the HLA DR genotypeDR3-DQB1*0201/DR4-DQB1*0302 (referred to as DR3/4 below) appear to be atthe highest risk for type 1 diabetes (see Noble, Valdes et al., 1996),incorporated herein by reference). These high-risk individuals haveabout a 1 in 15 chance of being affected with type 1 diabetes. Becauseof the strong effect of this genotype on the likelihood of type 1diabetes, the presence of the DR3/4 genotype could mask the contributionof IL4R alleles or haplotypes.

The distribution of IBD in families was stratified into two groups basedon the DR3/4 genotype of the children. The first group contains thefamilies in which one or both of the sibs are DR3/4 (“Either/both sibDR3/4”, n=119). The second group contains the families where neitherchild is DR3/4 (“Neither sib DR3/4”, n=137). The IBD distribution inthese subgroups is shown in Table 13. There was no statisticallysignificant departure from the expected distribution of IBD sharing inthe “either/both sib DR3/4” subgroup of families. There is astatistically significant departure from the expected distribution ofIBD sharing in the “neither sib DR3/4” subgroup of families (Table 13).This indicates that there is evidence for linkage of the IL4R loci toIDDM in the “neither sib DR3/4” families.

Association by AFBAC

Association of IL4R haplotypes with type 1 diabetes was assessed usingthe AFBAC (Affected Family Based Control) method (Thomson, G., 1995, AmJ Hum Genet 57:487-98). In essence, two groups of haplotypes, and thehaplotype frequencies in the groups, are compared with each other as ina case/control scheme of sampling. These two groups are the case(transmitted) and the control (AFBAC) haplotypes.

The case haplotypes, namely those transmitted to the affected children,were collected and counted as follows. For every pair of siblings,regardless of the status of the parents (homozygote or heterozygote) allfour transmitted chromosomes were counted. However, the haplotypes inthe two siblings in a pair are not independent of each other. The way tomake a statistically conservative and valid enumeration is to divide allcounts by two.

The control (AFBAC) haplotypes are those that are never transmitted tothe affected pair of children (Thomson, 1995). The AFBAC haplotypespermit an unbiased estimate of control haplotype frequencies. AFBACs canonly be determined from heterozygous parents, and furthermore, only whenthe parent transmits one haplotype to both children; the other,never-transmitted haplotype is counted in the AFBAC population. TheAFBAC population serves as a well-matched set of control haplotypes forthe study.

Table 14A shows the comparison of transmitted and AFBAC frequencies forall HBDI haplotypes that were observed at least five times in thecomplete sample set. Each row represents data on an individualhaplotype. However, in all 16 distinct haplotypes were observed in theHBDI data set, although some very rarely. The seven rarest haplotypesare grouped together in the “others” row. Each haplotype is listed bythe ordered set of alleles (namely, from SNPs 1-2-3-4-5-6-7-8) presentat each of the eight IL4R SNPs as described in Table 6. A “1” denotesthe presence of the reference allele, a “2” the presence of the variantallele for each SNP. The “reference” allele for each SNP is that presentin GenBank Accession X52425.1 as described in Table 6.

Tables 14B and 14C show the comparison of transmitted and AFBACfrequencies for all HBDI haplotypes seen in the “either/both sib DR3/4”and the “neither sib DR3/4” subgroups of families, respectively. Thesetables show that stratifying the families based on the DR3/4 genotype ofthe children permits the identification of haplotypes that areassociated with IDDM. In particular, in the “neither sib DR3/4” subgroupone haplotype (labeled “2 1 2 2 2 2 2 1”) is significantly underrepresented in the pool of transmitted chromosomes (P<0.005).

From the transmitted and AFBAC haplotype frequency information in Tables14B and 14C, one can derive by counting the frequencies of transmittedand AFBAC alleles. The locus-by-locus AFBAC analyses are shown in Tables15A and 15B.

The data present in Tables 15A and 15B show that there statisticallysignificant evidence, in the “neither sib DR3/4” subgroup of families,that alleles of SNPs numbers 3, 4 5, 6, and 7 are associated with IDDM.The evidence for association is especially strong for SNP #6. In the“either/both sib DR3/4” subgroup, there is the same trend of allelicassociation, although the trend does not quite reach statisticalsignificance.

Association by Haplotype-Based TDT

The TDT analysis can be utilized for determining the transmission (ornon-transmission) of 8-locus haplotypes from parents to affectedchildren, once the haplotypes have been inferred or assigned bymolecular means. Tables 16A, B, and C summarize the TDT results for theHBDI families. Each haplotype is listed by the ordered set of alleles(namely, from SNPs 1-2-3-4-5-6-7-8) present at each of the eight IL4RSNPs as described in Table 6. A “1” denotes the presence of thereference allele, a “2” the presence of the variant allele for each SNP.The “reference” allele for each SNP is that present in GenBank AccessionX52425.1 as described in Table 6. Table 16A counts informativetransmission events only to one child (the proband) per family, Table16B counts informative transmissions to the two primary affectedchildren per family, and Table 16C counts informative transmissions toall affected children. The 8-locus haplotype TDT results reachstatistical significance when all affected children (2 or more perfamily) are included.

The TDT analyses can be performed on families after stratifying for theDR3/4 genotype of the children. The summary of counts of informativetransmissions to the two primary affected children per family, in the“either/both sib DR3/4” and the “neither sib DR3/4” subgroups offamilies, are shown in tables 17A and 17B respectively. Each haplotypeis listed by the ordered set of alleles (namely, from SNPs1-2-3-4-5-6-7-8) present at each of the eight IL4R SNPs as described inTable 6. A “1 ” denotes the presence of the reference allele, a “2” thepresence of the variant allele for each SNP. The “reference” allele foreach SNP is that present in GenBank Accession X52425.1 as described inTable 6.

As presented above, there is significant evidence of linkage of IDDM toIL4R in the “neither sib DR3/4” subgroup. The data in Table 17B indicatethat there is significant evidence of association of IL4R haplotypes toIDDM, in the presence of this linkage. In particular, in the “neithersib DR3/4” subgroup one haplotype (labeled “2 1 2 2 2 2 2 1”) issignificantly under-transmitted to affected children.

9 EXAMPLE 3 Association of IL4R with Type 1 Diabetes in Filipino Samples

This example demonstrates the association of IL4R SNPs with type 1diabetes in a Filipino population.

As used in this section, “patients” refers to individuals with thedisease, namely individuals with type 1 diabetes and “controls” refersnormal individuals, those without the disease.

Ninety patients (n=90) were selected for this study from amongst theFilipino population. The patients included in the study were affected bytype 1 diabetes as defined by the recent ADA classification (the ExpertCommittee on the Diagnosis and Classification of Diabetes Mellitus1997). The patients were born in the Philippines and all had twoFilipino parents. These patients had been characterized for C-peptidelevels below 0.3mmol/l and for autoantibodies to islet cell autoantigens(Medici et al., 1999, Diabetes Care, 9:1458-62). Samples were alsocollected from ninety-four Filipino normal subjects without a familyhistory for diabetes. This was the control group. All patients andcontrols were from the southern region of Luzon, Philippines. The studywas approved by the local Ethics Committee and informed consent wasgiven by patients. In addition, independent samples from a previousstudy of HLA class II loci in Filipinos (Bugawan et al., 1994, Genetics,54:331-340) originating from the same region were used, following astatistical test of heterogeneity, to supplement the control samples.These comprised a total of 194 chromosomes taken from family andindividual samples.

The individuals were genotyped as described above. The genotypes of theaffected and unaffected individuals are shown in Table 4 (SEQ ID NO:20-24). Both the actual numbers and the frequencies are provided foreach genotype. The data (Table 5) confirm the presence of an associationof IL4R SNP variants with type 1 diabetes.

Statistical Methods & Algorithms

Allele and haplotype frequencies between groups were compared using thez-test. Haplotype compositions and frequencies were estimated from thegenotype data using the EM algorithm in the Arlequin program (L.Excoffier, University of Geneva, CH) (Excoffier et al., 1995, Mol BiolEvol, 12:921-7; Slatkin et al., 1996, Heredity, 76:377-83).

Results

The wildtype allele frequencies for each of the eight IL4R SNPs in theFilipino control and diabetic groups are shown in Table 18. Table 18provides evidence that the allele frequencies for SNPs #3 and 4 aresignificantly different between the two groups, and suggests anassociation to IDDM.

It is also possible to infer and construct the multi-locus IL4Rhaplotypes in the Filipino subjects, either computationally byMaximum-likelihood estimation (MLE), or by using molecular haplotypingmethods described previously. Table 19 lists the five most frequentcomputationally estimated haplotypes and their frequencies in theFilipino diabetics and controls, and presents the significance of thedifferences in frequencies. Each haplotype is listed by the ordered setof alleles (namely, from SNPs 1-2-3-4-5-6-7-8) present at each of theeight IL4R SNPs as described in Table 6. A “1” denotes the presence ofthe reference allele, a “2” the presence of the variant allele for eachSNP. The “reference” allele for each SNP is that present in GenBankAccession X52425.1 as described in Table 6.

Table 20 lists the observed haplotypes as derived and inferred bymolecular haplotyping; the unambiguous seven-locus haplotypes (SNP#1allele not shown, as indicated by the “x”) are compiled. Each haplotypeis listed by the ordered set of alleles (namely, from SNPs1-2-3-4-5-6-7-8) present at each of the eight IL4R SNPs as described inTable 6. A “1” denotes the presence of the reference allele, a “2” thepresence of the variant allele for each SNP. The “reference” allele foreach SNP is that present in GenBank Accession X52425.1 as described inTable 6. Tables 18 and 19 both provide evidence of a statisticallysignificant difference in the frequency of one or more haplotypesbetween the Filipino control and diabetic populations, and support thepresence of an association of IL4R to IDDM. In particular, the haplotype(labeled “x 1 2 2 2 2 2 1”) is significantly under represented in theFilipino diabetics group.

10. EXAMPLE 4 Association of IL4R, IL4 and IL13 SNPs with Type 1Diabetes in Filipino Samples

This example demonstrates the association of IL4R, IL4 and IL13 SNPswith type 1 diabetes in the same Filipino population as described above,in Example 3.

As used in this section, “patients” refers to individuals with thedisease, namely individuals with type 1 diabetes and “controls” refersnormal individuals, those without the disease.

The individuals were genotyped as described above.

Individual SNPs

The distributions of alleles at the individual SNPs in the IL4R locus(n=10), the IL4 locus (n=1) and the IL13 locus (n=4) among patients andcontrols are shown in Table 21. Linkage disequilibrium patterns wereestimated using maximum likelihood approaches from individual genotypedata from unrelated individuals (Slatkin and Excoffier, 1996 Heredity76:377:383). The patterns of pairwise linkage disequilibrium (LD) forthese SNPs inferred among the control population are shown in Tables 22and 23. Among the individual IL4R SNPs, three (E375A, L389L, and C406R)showed a nominally significant association with type 1 diabetes whilethe variant allele at two additional SNPs (I50V, p=0.062 and S478G,p=0.064) was decreased among patients (Table 21).

The two promoter SNPs were not significantly associated with type 1diabetes, although the variant allele of the −3223 SNP was slightlyincreased among patients (OR=1.45, p=0.10). With the exception of thispromoter SNP and the I50V SNP, with which it is in strong LD, thevariant allele at each SNP was under represented among patients. Some ofthe polymorphic amino acid residues in this chain appear to bebiologically important and affect IL-4 receptor signaling (Kruse et al.,1999, Immunology, 96:365-71).

Of the 10 IL4R SNPs typed, the L389L SNP showed the strongestassociation with type 1 diabetes in this population, with significantlylower frequencies among patients than controls (OR=0.34; p=0.001).Without being bound by theory, it is believed that because this is asilent (synonymous) polymorphism, it is unlikely that this SNP isresponsible for the observed protective effect for type 1 diabetes. ThisSNP is in very strong LD (Table 22) with the nonsynonymous flanking SNPs(E375A, C406R, S478P and Q551R) and that these SNPs all show a trendtoward protection (negative association). The L389L SNP is also instrong negative LD with the −3223 promoter SNP (Table 22).

In the comparison of genotypes at the individual IL4R SNPs (Table 24),the protective effect is dominant in that the heterozygote for IL4R 389has an OR=0.29. Among the individual SNPs on chromosome 5q31, only thevariant alleles at the two IL13 promoter SNPs were increased amongpatients (OR=1.58 and p =0.05 for −1512 and OR=1.49 and p=0.12 for−1112) (Table 21) When genotype frequencies are compared, however, theIL13 R 1000Q showed a nominally significant association in thispopulation (p=0.03; Table 24). These data suggest that the varianthomozygote, but not the heterozygote, may be at increased risk for type1 diabetes. In Table 24, each haplotype is listed by the ordered set ofalleles (namely, from SNPs 1-2-3-4-5-6-7-8) present at each of the eightIL4R SNPs as described in Table 6. The letters refer to the actualallele (nucleotide) present, as described in Table 6.

Haplotypes

IL4R

IL4R haplotypes were estimated based on an expectation-maximization (EM)method (Excoffier et al., 1995, Mol. Biol. Evol., 12:921-927.) and weredirectly determined by molecular haplotyping methods, described inExample 4. The molecular haplotyping method allowed the unambiguousassignment of phase for 7 IL4R SNPs (C676T, A1374C, G1417T, T1466C,T1682C, A1902G and T2531C). Using molecular haplotyping of these 7 SNPs,7 different IL4R haplotypes were determined in this population and theirfrequencies among patients and controls compared (Table 25A). In Table25A each haplotype is listed by the ordered set of alleles (namely, fromSNPs 2-3-4-5-6-7-8) present at seven of the eight IL4R SNPs as describedin Table 6. The letters refer to the actual allele (nucleotide) present,as described in Table 6.

One specific haplotype (CCTCCGT)) was significantly under representedamong patients (OR=0.4, p=0.013). This same haplotype was also found tobe protective (significant negative association), by the TDT methods, inthe HBDI families, as described in Example 2. This protective effect hasthus been observed in two different populations and in two differentstudy designs, namely case/control (Filipino) and TDT, in addition tothe biological plausibility (i.e., functional consequences) of theseSNPs. This strongly suggests that variants of the IL4R moleculeinfluence the susceptibility to type 1 diabetes. In particular, thisspecific haplotype of IL4R appears to confer a dominant protectiveeffect.

In the HBDI families, stratification based on the highest risk HLAgenotype (HLA-DRB1*0301-DQB1*0201/HLA-DRB1 *04-DQB1*0302) was necessaryto demonstrate the protective effect of the IL4R haplotype. Asignificant negative association was found only among those families inwhich neither affected sib was DR3/4, presumably because the effect ofthe IL4R polymorphism was relatively modest compared to the riskconferred by this high risk HLA genotype, which confers a disease riskhigher than DR3/3 or DR4/4 homozygotes. Among Filipinos, a significantprotective effect of a specific IL4R haplotype was observed withoutstratification (Table 25). Without being bound by theory, this mayreflect the absence among Filipinos of a higher risk associated withDR3/4 than with DR3/3 or DR4/4 genotypes. The lack of the “DR3/4effect,” well-established by many studies of Caucasian type 1 diabetes,can be attributed among Filipinos to the differing patterns of linkagedisequilibrium of DQB1 alleles with DRB1*04 alleles between Asians andCaucasians.

The molecular haplotyping approach used in this example did not assignphase for the two promoter SNPs and the I50V SNP in the IL4R locus.Consequently, the EM approach was used to estimate frequencies for10-SNP haplotypes for these 3 individual SNPs and the 7-SNP haplotypepreviously determined by molecular methods (Table 25B). In Table 25B,each haplotype is listed by the ordered set of alleles (namely, fromSNPs 1-2-3-4-5-6-7-8-9-10) present at each of the ten IL4R SNPsdescribed in Table 21 (SNPs in order:C(−3223)T-C(−1914)T-I50V-N142N-E375A-L389L-C406R-S478P-Q551R-S761P). Theletters refer to the actual allele (nucleotide) present, as described inTable 21.

Of the 17 10-SNP haplotypes with an estimated frequency >1%, in eithergroup, only one 10-SNP haplotype containing the protective 7-SNPhaplotype (H5A or CCA-CCTCCGT) appeared strongly negatively associated(OR=0.0; 95%CI [0-0.5]; p=0.001) with disease. Interestingly, the otherhaplotype which contained the same 7-SNP haplotype (H5B or CTA-CCTCCGT)was not significantly associated with disease (OR=0.66 p=0.33). Thissuggests that a specific combination of IL4R promoter SNPs with aparticular coding sequence variant contributes to the risk for type 1diabetes.

IL4 and IL13

The IL4 and the IL13 SNPs are in strong LD (Table 23). The estimatedfrequencies for these 5-SNP haplotypes were compared among patients andcontrols (Table 26). In Table 26, each haplotype is listed by theordered set of alleles present at the one IL4 SNP and the four IL13 SNPsas described in Table 21. The order is IL4 C(−524)T-IL13 A(−1512)C-IL13C(−1112)T-IL13 intron3-IL13 R110Q. The letters refer to the actualallele (nucleotide) present, as described in Table 21.

The overall distributions were different (p=0.005) and one haplotype,TCTTA, was strongly associated with type 1 diabetes (OR=3.47, p=0.004).Two other haplotypes showed a nominally significant association (p=0.02and 0.03). One surprising observation was that the IL13 haplotype CTTAappeared to be associated with disease only in combination with the Tallele at the IL4 −524 promoter SNP because the CCTTA haplotype showedno disease association. These data could reflect LD between theassociated 5-SNP haplotype with some nearby causal gene or suggest thata particular combination of a promoter variant at IL4 and promoter andcoding variants at IL13 are responsible for an elevated type 1 diabetesrisk (gene-gene interaction).

13. EXAMPLE 7 Dependence of Risk for Type 1 Diabetes Conferred by IL4RSNPs on Genotype at IL4 and IL13 in Filipino Samples: Evidence forEpistasis

This example demonstrates epistasis, the interaction between SNPs on theIL4R locus on chromosome 16 and those on the IL4 and IL13 loci onchromosome 5.

Because IL4 and IL13 both serve as ligands for a receptor composed, inpart, of the IL4R alpha chain, there is a likelihood of gene-geneinteractions between polymorphisms in the IL4R locus on chromosome 16p11and the five SNPs in the IL4 and IL13 loci on chromosome 5q31. In oneapproach, the statistical independence for genotypes at the 10 IL4R SNPsand the genotypes at each of the IL4 and IL13 SNPs (Table 27) wasexamined. Gene by gene interactions at SNPs in different genes wereevaluated by assessing whether the genotype frequencies at unlinked lociwere independent (i.e., the IL13 and IL4 SNPs on chromosome 5 and theIL4R SNPs on chromosome 16) among patients. These analyses were done foreach pair of unlinked SNPs carrying out a chi-square test in contingencytables with marginals defined by genotype counts either in patients orcontrols. The chi-square values and the corresponding degrees of freedomfor each IL4R SNP comparisons were summed and p-value of the sum ofchi-squares computed.

No deviation from independence was found for these SNPs among controlsbut a significant deviation was found for the IL4 −524 promoter SNP(p=0.001) and the IL13 intron 3 SNP (p=0.019) among patients.

To assess whether the effect on type 1 diabetes susceptibility due toIL4R SNPs was modified by the IL4 or the IL13 SNPs, epistasis wasmodeled using a logistic regression model (see below). For each of thefive IL4 and IL13 SNPs, we tested whether the effect of the combinedIL4R SNP genotypes on type 1 diabetes susceptibility differed dependingon the IL4 and IL13 SNPs. The results (Table 28) indicate that there isindeed an epistatic interaction between the IL4R genotypes and IL4 andIL13 genotypes. To address the issue of multiple comparisons, we carriedout permutation analysis on this test. In 22/200 permuations one or moreof the 5-SNP tests showed a p<0.035, in 13/200 one or more of the SNPtests had p<0.035 and another one had p<0.075. In 9/200 one or more ofthe 5-SNP tests showed a p<0.035, another had a p<0.075 and another hada p<0.135. Thus, the pattern observed in Table 28 has a probability ofp<0.045. The conclusion from this is that the epistatic interactionobserved between the IL4 and IL13 SNPs and the IL4R genotypes isstatistically significant indicating that, in this data set, thegenotypes in the IL4, IL13 region affect the genetic susceptibility totype 1 diabetes conferred by IL4R.

To illustrate this interaction, the odds ratios for individual IL4R SNPsas a function of the IL4 and IL13 SNP genotype were also calculated. Thedifferences among the Odds Ratios were greatest for the IL4R -3223 SNPand the four IL13 SNPs. The odds ratios with the 95% confidenceintervals and the p values from the stratified contingency tableanalyses are shown in FIG. 2. FIG. 2 shows the Odds ratios, patient andcontrol counts of specific IL4R and IL4, IL13 genotypes. FIG. 2A showsthe interaction of IL4R Ala375Glu with and IL4 −524 (promoter). FIG. 2Bshows the interaction of IL4R Gln551Arg with IL13 A-1512C. FIG. 2C showsthe interaction between IL4R C-3223T and IL13 A-1512C and FIG. 2D showsthe interaction between IL4R C-3223T and IL13 C-1112T. The p-value forthe association of each genotype combination is shown above each oddsratios bar.

The most striking observation was that the IL4R −3223 SNP CT genotypehad an OR of 8.55 (95% CI=1.05, 69.8) when present with the TT IL13−1112 genotype and an OR=0.53 (95% CI=0.29, 0.98) when present with theCC genotype. Without being bound by theory, the observation of aninteraction between polymorphisms in the IL13 and IL4 genes andpolymorphism in the gene encoding the receptor for the products of thesetwo genes represents an interesting and biologically plausiblehypothesis that, given the multiple comparisons, requires furthertesting. A recently published study of asthma patients reported agene-gene interaction between IL4R and IL13 in the determination ofserum IgE levels (Howard et al., 2002, Am J Hum Genet, 70:230-6).

As described above, the IL4R association data obtained in this Filipinocase control study indicate that the 7-SNP haplotype, composed primarilyof variant alleles at these SNPs, confers dominant protection to type 1diabetes (Table 25A), consistent with our recent observations, based onTDT and AFBAC analysis in a set of multiplex Caucasian families (theHBDI registry). The replication of this observation in two differentpopulations and in two different study designs strengthens thisinference. The analysis of 10-SNP IL4R haplotypes among Filipinossuggests that a specific promoter variant in combination with specificcoding sequence variants may be responsible for the observed protection(Table 25B). Several recent studies have shown that the reference orwild-type allele at several of these IL4R SNPs is associated with atopicasthma and increased IgE levels (Howard et al., 2002, Am J Hum Genet,70:230-6, Sandford et al., 2000, J Allergy Clin Immunol, 106:135-40).Thus, it appears that the same alleles at IL4R SNPs confer an increasedrisk to a canonical Th1 (type 1 diabetes) and a Th2 disease (atopicasthma). Without being bound by theory, these associations argue againstan effect on Th1/Th2 balance mediated by polymorphism in the IL4R geneand suggest instead that this polymorphism may influence some aspect ofimmune regulation and homeostasis in Th1 and Th2 pathways and possibly,B cell activation. Conceivably, the observed patterns of diseaseassociation reflect the effect of IL4R polymorphisms on the balancebetween the activation of Th1 and Th2 cells and that of T regulatorycells. In conclusion, the extent of risk for type 1 diabetes may bedetermined by specific combinations of variants at the IL4R locus and atthe genes encoding its two ligands, IL4 and IL13.

Calculations Performed to Achieve the Results of Table 28

For each IL4R SNP, the homozygote genotype with the highest odds ratiowas given a value of 2, the heterozygote was given a value of 1, theother homozygote was 0. A logistic regression was carried out on nineIL4R polymorphisms (S761P did not show the variant) in this way and anew numerical variable “il4r” was derived given by:il4r=a ₁ G ⁻³²²³ +a ₂ G ⁻¹⁹¹⁴ +a ₃ G ₅₀ +a ₄ G ₁₄₂ +a ₅ G ₃₇₅ +a ₆ G ₃₈₉+a ₇ G ₄₀₆ +a ₈ G ₄₇₈ +a ₉ G ₅₅₁where Gi denotes the genotype (0, 1 or 2) at the ith position and ajdenotes the coefficient fitted by logistic regression. The coefficientsfitted by the regression were a1=0.368; a2=0.053; a3=0.37; a4=0.061;a5=0.66; a6=1.08; a7=0.57; a8=0.54 and a9=0.22. Epistasis was thentested independently for each of the five chromosome 5 SNPs by fittingthe following logistic regression model: P(T1DM)=exp(X)/(1+exp(X)) WhereX=C+β₁il4r+β₂G_(chr5i)+β₃(il4r−G_(chr5i)) and G_(chr5i) is the genotypeof one of the chromosome 5 SNPs (values 0, 1 or 2).

Permutation Analysis

Because five different SNPs were compared, it was important to correctfor multiple tests. However, a Bonferroni or a Dunn-Sidak correction wasnot appropriate since the IL4 and IL13 SNPs are not independent (seeTable 23). Therefore permutation analysis was carried out, keepingconstant the patient and control genotype frequencies, but permutatingthe IL4 and IL13 genotypes and the IL4R genotypes within the patient andwithin the control groups separately. In this way, only the epistaticinteraction between the two genetic regions was tested and not theindividual IL4 or IL13 and IL4R genetic associations. 200 permutationswere carried testing for epistasis at all five chromosome 5 SNPs eachtime. Analyses were carried out using S-Plus version 6.0 Professional(Insightful Corporation).

Various embodiments of the invention have been described. Thedescriptions and examples are intended to be illustrative of theinvention and not limiting. Indeed, it will be apparent to those ofskill in the art that modifications may be made to the variousembodiments of the invention described without departing from the spiritof the invention or scope of the appended claims set forth below.

All references cited herein are hereby incorporated by reference intheir entireties. TABLE 3 Variant Probe Probe Sequence Seq ID No: titer(μl) A398 DBM0081P CCACACGTGTATCCCTGAGAA 3 1.0 398G DBM0082PTCTCAGGGACACACGTGTG 4 4.0 C676 DBM0172P TGGAGTGAAAACGACCCGGCAG 5 1.0676T DBM0073P CTGCCGGGTCATTTTCGCTCC 6 1.0 A1374 DBM0043RCGAGGGAAGGGAGGGCATTGTG 7 1.0 1374C DBM0139P AGGGAAGGGCGGGCATTGT 8 4.0G1417 DBM0124P CTCTCCGAGCAGGTCCAGG 9 1.0 1417T DBM0046RCTCCTGGACCTTCTCGGAGAGG 10 1.0 T1466 DBM0076P AAGGTGGAAGAAGGCATGACTCC 111.0 DBM0132P AAGGTGGGAGAAGACATGACTCC 12 1.0 1466C DBM0171PGGAGTCACGTCTTCTCCTACCTT 13 2.0 T1682 DBM0157P TGGCTCAGAGAGTTGCTGAAGC 142.0 1682C DBM0094P TTCAGCAACCCCCTGAGCC 15 2.0 A1902 KW86AGTGGCTATCAGGAGTTTGT 16 1.6 1902G KW85 AGTGGCTATCGGGAGTTTGT 17 0.8 T2531DBM0080P CTCTTCTCTGAGATGCCCGAG 18 0.5 2531C DBM0048RCCTCGGGCATCCCAGAGAAGAG 19 0.5

TABLE 4 Computationally estimated haplotype frequencies compared betweenFilipino controls and diabetics. Seq MLE MLE COUNT: COUNT: Total exp exphaplotype ID No: label frequency frequency O.R. control diabetics countcontrol diabetic chi-square P-value ACATTTAT 20 H-1 0.318 0.329 1.1 59.758.5 118.2 60.7 57.5 0.033 0.855 ACAGTTGT 21 H-6 0.074 0.074 1.0 14.013.2 27.2 14.0 13.2 0.000 0.987

22 H-3 0.145 0.058 0.4 27.3 10.3 37.6 19.3 18.3 6.727 0.009 GCAGTTAT 23H-2 0.390 0.441 1.2 73.3 78.5 151.8 78.0 73.8 0.582 0.445 GCCGCCGT 24H-10 0.032 0.051 1.6 6.0 9.0 15.0 7.7 7.3 0.776 0.378 n = 188 178 366overall 8.118 0.0987

TABLE 5 Affected Genotypes Control Genotypes SNP (n = 89) (n = 94) A398GAA = 21 AG = 40 GG = 28 AA = 32 AG = 41 GG = 21 (50 I/V) (0.236) (0.449)(0.315) (0.340) (0.436) (0.223) C676G CC = 89 CC = 92 CG = 2 (142 N/N)(1) (0.979) (0.021) A1374C AA = 70 AC = 17 CC = 2 AA = 55 AC = 30 CC = 3(375 E/A) (0.787) (0.191) (0.023) (0.630) (0.341) (0.034) G1417T GG = 78GT = 10 TT = 1 GG = 63 GT = 29 TT = 2 (389 L/L) (0.876) (0.112) (0.011)(0.670) (0.309) (0.022) T1466C TT = 70 TC = 17 CC = 2 TT = 60 TC = 32 CC= 2 (406 C/R) (0.787) (0.191) (0.023) (0.638) (0.340) (0.022) T1682C TT= 70 TC = 17 CC = 2 TT = 61 TC = 31 CC = 2 (478 S/P) (0.787) (0.191)(0.023) (0.649) (0.330) (0.022) A1902G AA = 50 AG = 35 GG = 3 AA = 50 AG= 36 GG = 8 (551 Q/R) (0.562) (0.393) (0.034) (0.532) (0.383) (0.085)T2531C TT = 89 TT = 94 (761 S/P) (1) (1)

TABLE 6 SNPs detected Genbank SNP # LOCUS SNP Variation Access # 1 IL4RA398G I50V X52425 2 IL4R C676T N142N X52425 3 IL4R A1374C E375A X52425 4IL4R G1417T L389L X52425 5 IL4R T1466C C406R X52425 6 IL4R T1682C S478PX52425 7 IL4R A1902G Q551R X52425 8 IL4R T2531C S761P X52425

TABLE 7 Amplicon primers and lengths Amplicon Seq Amplicon Seq AmpliconLeft Primer ID Right Primer ID SNP # size, bp Name Left primer sequenceNo: Name Right primer sequence No: 1 114 RR192B CAGCCCCTGTGTCTGCAGA 25RR193B GTCCAGTGTATAGTTATCCGCACTGA 31 2 163 DBM0177B CTGACCTGGAGCAACCCGTA26 DBM0178B ACTGGGCCTCTGCTGGTCA 32 3, 4, 5 228 DBM0023BATTGTGTGAGGAGGAGGAGGAGGTA 27 DBM0022B GTTGGGCATGTGAGCACTCGTA 33 6 129DBM0097B CTCGTCATCGCAGGCAA 28 DBM0098B AGGGCATCTCGGGTTCTA 34 7 198RR200B GCCGAAATGTCCTCCAGCA 29 RR178B CCACATTTCTCTGGGGACACA 35 8 177DBM0112B CCGGCCTCCCTGGCA 30 DBM0071B GCAGACTCAGCAACAAGAGG 36

TABLE 8 Hybridization probes and titers Variant allele Seq WT alleleVariant Seq probe SNP WT allele WT allele ID probe allele Varient alleleprobe ID titer # Probe name probe sequence No: titer (μM) probe namesequence No: (μM) 1 DBM0081P CCACACGTGTATCCCTGAGAA 37 1.0 DBM0082PTCTCAGGGACACACGTGTG 46 4.0 2 DBM0172P TGGAGTGAAAACGACCCGGCAG 38 1.0DBM0073P CTGCCGGGTCATTTTCGCTCC 47 1.0 3 DBM0043RC GAGGGAAGGGAGGGCATTGTG39 1.0 DBM0139P AGGGAAGGGCGGGCATTGT 48 4.0 4 DBM0124PCTCTCCGAGCAGGTCCAGG 40 1.0 DBM0046RC TCCTGGACCTTCTCGGAGAGG 49 1.0 5DBM0076P + AAGGTGGAAGAAGGCATGACTCC 41 1.0 DBM0171PGGAGTCACGTCTTCTCCTACCTT 50 2.0 DBM0132P AAGGTGGGAGAAGACATGACTCC 42 1.0 6DBM0157P TGGCTCAGAGAGTTGCTGAAGC 43 2.0 DBM0094P TTCAGCAACCCCCTGAGCC 512.0 7 KW86 AGTGGCTATCAGGAGTTTGT 44 1.6 KW85 AGTGGCTATCGGGAGTTTGT 52 0.88 DBM0080P CTCTTCTCTGAGATGCCCGAG 45 0.5 DBM0048RC CTCGGGCATCCCAGAGAAGAG53 0.5

TABLE 9 Allele Frequency Of wildtype allele in HBDI founders WT AlleleSNP # Frequency 1 0.53 2 0.90 3 0.89 4 0.89 5 0.89 6 0.83 7 0.79 8 0.99

TABLE 10 Pairwise Linkage Disequilibrium Measures

TABLE 11A Results of single locus TDT analysis, to the 282 probands onlyTransmissions of Non-transmissions McNemar chi- WT allele to of WTallele to Total observed squared SNP # affected (T) affected (NT)Transmissions (T + NT) statistic Significance 1 144 138 282 0.128 N.S. 255 39 94 2.723 N.S. 3 62 46 108 2.370 N.S. 4 62 46 108 2.370 N.S. 5 6146 107 2.103 N.S. 6 90 67 157 3.369 N.S. 7 103 81 184 2.630 N.S. 8 5 712 0.333 N.S.

TABLE 11B Results of single locus TDT analysis, to 564 primary affectedand 26 additional children Non- Transmissions of transmissions of Totalobserved McNemar WT allele to WT allele to Transmissions chi-squaredTransmission SNP # affected (T) affected (NT) (T + NT) statisticSignificance T/(T + NT) 1 311 280 591 1.626 N.S. 2 107 90 197 1.467 N.S.3 130 98 228 4.491 P < 0.05 57.0% 4 130 98 228 4.491 P < 0.05 57.0% 5128 98 226 3.982 P < 0.05 56.6% 6 189 149 338 4.734 P < 0.05 55.9% 7 212180 392 2.612 N.S. 8 12 13 25 0.040 N.S.

TABLE 12 Allele specific PCR primers and amplicon lengths SNP detectedOther SNPs Wildtype Allele- allele- Amplicon spanned by Common SpecificPrimer specifically Size. bp amplicon Primer Name Common PrimerSequences Seq ID No: Sequence 3 678 4, 5, 6, 7 RR178BCCACATTTCTCTGGGGACACA 54 DBM0149B 7 639 3, 4, 5, 6 DBM0023BATTGTGTGAGGAGGAGGAGGAGGTA 55 DBN9155B 6 413 3, 4, 5 DBM0023BATTGTGTGAGGAGGAGGAGGAGGTA 56 DBM0194B SNP detected Variant Allele-allele- Wildtype Allele-specific Primr Specific Primer VariantAllele-specific Primer specifically Sequence Seq ID No: Name SequencesSeq ID No: 3 TTCCAGGAGGGAAGGGA 57 DBM0150B TTCCAGGAGGGAAGGGC 60 7CACCGCATGTACAAACTCCT 58 DBM0156B CACCGCATGTACAAACTCCC 61 6GGTGACTGGCTCAGGGA 59 DBM0195B GGTGACTGGCTCAGGGG 62

TABLE 13 IBD Distributions for IL4R haplotypes Group (n) IBD = 2 IBD = 1IBD = 0 P-value All families (256) 30.7% 47.9% 21.5% N.S. Either/bothsib DR3/4 (119) 26.1% 50.4% 23.5% N.S. Neither sib DR3/4 (137) 34.7%45.6% 19.7% 0.03 Expectation   25%   50%   25%

TABLE 14A Haplotype transmissions by AFBAC method: all families CountsObserved Frequency Chi-squared IL4R 8-locus Haplotype Transmitted AFBACTotal Transmitted AFBAC Statistic p-value 1 1 1 1 1 1 1 1 259.5 109.5369 46.3% 41.8% 0.82 0.36 2 1 1 1 1 1 1 1 170.5 71.5 242 30.4% 27.3%0.60 0.44 2 1 2 2 2 2 2 1 29.5 23.5 53 5.3% 9.0% 3.79 0.05 2 2 1 1 1 1 11 23.5 13 36.5 4.2% 5.0% 0.24 0.63 1 1 2 2 2 2 2 1 23 12.5 35.5 4.1%4.8% 0.18 0.67 2 2 1 1 1 2 2 1 20.5 14 34.5 3.7% 5.3% 1.20 0.27 1 1 1 11 1 2 1 19 9 28 3.4% 3.4% 0.00 0.98 2 1 1 1 1 1 2 1 3.5 3 6.5 0.6% 1.1%0.61 0.43 2 2 1 1 1 2 2 2 4.5 2 6.5 0.8% 0.8% 0.00 0.95 Others 6.5 410.5 1.2% 1.5% 0.19 0.67 TOTAL 560 262 822 7.64 0.94

TABLE 14B Haplotype transmissions by AFBAC method: “Either/both sibDR3/4” subgroup of familes Counts Observed Frequency Chi-squared IL4R8-locus Haplotype Transmitted AFBAC Total Transmitted AFBAC Statisticp-value 1 1 1 1 1 1 1 1 254 50.5 304.5 46.7% 41.6% 0.57 0.45 2 1 1 1 1 11 1 156 30 186 28.7% 24.7% 0.56 0.45 2 1 2 2 2 2 2 1 35 11 46 6.4% 9.1%0.99 0.32 2 2 1 1 1 1 1 1 24 7 31 4.4% 5.8% 0.39 0.53 1 1 1 1 1 1 2 1 206 26 3.7% 4.9% 0.40 0.52 1 1 2 2 2 2 2 1 18 7 25 3.3% 5.8% 1.59 0.21 2 21 1 1 2 2 1 18 4 22 3.3% 3.3% 0.00 0.99 2 2 1 1 1 2 2 2 8 2 10 1.5% 1.6%0.02 0.89 2 2 1 1 1 2 2 2 8 1 9 1.5% 0.8% 0.31 0.58 2 1 1 1 1 1 2 1 1 23 0.2% 1.6% 4.71 0.03 2 1 1 1 1 2 2 1 2 1 3 0.4% 0.8% 0.46 0.50 TOTAL544 121.5 665.5 10.00 0.44

TABLE 14C Haplotype transmissions by AFBAC method: “neither sib DR3/4”subgroup of families Counts Observed Frequency Chi-squared IL4R 8-locusHaplotype Transmitted AFBAC Total Transmitted AFBAC Statistic p-value 11 1 1 1 1 1 1 267 62.5 329.5 45.7% 40.7% 0.68 0.409 2 1 1 1 1 1 1 1 18543 228 31.7% 28.0% 0.53 0.467 2 1 2 2 2 2 2 1 24 15.5 39.5 4.1% 10.1%8.14 0.004 1 1 2 2 2 2 2 1 30 6 36 5.1% 3.9% 0.38 0.540 2 2 1 1 1 2 2 123 10 33 3.9% 6.5% 1.80 0.179 2 2 1 1 1 1 1 1 23 7.5 30.5 3.9% 4.9% 0.260.607 1 1 1 1 1 1 2 1 17 5 22 2.9% 3.3% 0.05 0.825 2 1 1 1 1 1 2 1 8 1 91.4% 0.7% 0.51 0.473 2 2 1 1 1 2 2 2 3 1 4 0.5% 0.7% 0.04 0.837 1 1 1 11 2 2 1 2 1 3 0.3% 0.7% 0.29 0.593 2 1 2 2 1 1 2 1 2 1 3 0.3% 0.7% 0.290.593 TOTAL 584 153.5 737.5 12.97 0.226

TABLE 15A SNP by SNP allele transmissions by AFBAC method: “either/bothsib DR3/4” subgroup of familes Transmitted (n = 540) AFBAC (n = 121.5)allele counts variant allele allele counts variant allele Chi-squaredp-value SNP # wt variant frequency wt allele variants frequencyStatistic (df = 1) 1 296 244 45.2% 64.5 57 46.9% 0.12 0.730 2 490 509.3% 107.5 14 11.5% 0.58 0.446 3 485 55 10.2% 103.5 18 14.8% 2.17 0.1414 485 55 10.2% 103.5 18 14.8% 2.17 0.141 5 485 55 10.2% 103.5 18 14.8%2.17 0.141 6 457 83 15.4% 96.5 25 20.6% 1.97 0.161 7 436 104 19.35 88.533 27.2% 3.77 0.052 8 532 8 1.5% 119.5 2 1.6% 0.02 0.893 TOTAL

TABLE 15B SNP by SNP allele transmissions by AFBAC method: “neither sibDR3/4” subgroup of familes Transmitted (n = 588) AFBAC (n = 158.5)allele counts variant allele allele counts variant allele Chi-squaredp-value SNP # wt variant frequency wt allele variant frequency Statistic(df = 1) 1 320 268 45.6% 77.5 81 51.1% 1.53 0.216 2 539 49 8.3% 139 19.512.3% 2.36 0.124 3 529 59 10.0% 132 26.5 16.7% 5.50 0.019 4 529 59 10.0%132 26.5 16.7% 5.50 0.019 5 534 54 9.2% 136 22.5 14.2% 3.41 0.065 6 50286 14.6% 121 37.5 23.7% 7.38 0.007 7 475 113 19.2% 115 43.5 27.4% 5.100.024 8 584 4 0.7% 157.5 1 0.6% 0.00 0.946

TABLE 16A TDT analysis of 8-loocus haplotypes to probands only. TOTALtransmitted to NOT transmitted TRANSMISSIONS McNemar chi- P-valuehaplotype affected to affected OBSERVED squared statistic (1 df) %TRANSMISSION 11111111 140 131 271 0.299 0.585 51.7% 21111111 130 105 2352.660 0.103 55.3% 21222221 27 39 66 2.182 0.140 40.9% 22111111 19 28 471.723 0.189 40.4% 11222221 19 24 43 0.581 0.446 44.2% 22111221 17 23 400.900 0.343 42.5% 11111121 18 15 33 0.273 0.602 54.5% 22111222 6 5 110.091 0.763 54.5% 21111121 1 5 6 2.667 0.102 16.7% 11111221 1 3 4 1.0000.317 25.0% 11221221 2 0 2 2.000 0.157 100.0% 21221121 1 1 2 0.000 1.00050.0% 21221221 0 2 2 2.000 0.157 0.0% 11221111 0 1 1 1.000 0.317 0.0%11111222 1 0 1 1.000 0.317 100.0% total 382 382 764

TABLE 16B TDT analysis of 8-loocus haplotypes to two primary affectedchildren per family. TOTAL transmitted to NOT transmitted TRANSMISSIONSMcNemar chi- P-value haplotype affected to affected OBSERVED squaredstatistic (1 df) % TRANSMISSION 21222221 52 80 132 5.939 0.015 39.4%21111111 245 225 470 0.851 0.356 52.1% 11111111 283 259 542 1.063 0.30352.2% 22111111 43 51 94 0.681 0.409 45.7% 11221221 3 1 4 1.000 0.31775.0% 11222221 44 42 86 0.047 0.829 51.2% 22111221 37 43 80 0.450 0.50246.3% 11111121 36 30 66 0.545 0.460 54.5% 22111222 11 11 22 0.000 1.00050.0% 21111121 3 9 12 3.000 0.083 25.0% 21221121 2 2 4 0.000 1.000 50.0%21221221 0 4 4 4.000 0.046 0.0% 11111221 4 4 8 0.000 1.000 50.0%11221111 0 2 2 2.000 0.157 0.0% 11111222 1 1 2 0.000 1.000 50.0%

TABLE 16c TDT analysis of 8-loocus haplotypes to all affected children.TOTAL transmitted to NOT transmitted TRANSMISSIONS McNemar chi- P-valuehaplotype affected to affected OBSERVED squared statistic (1 df) %TRANSMISSION 11111111 295 272 567 0.933 0.334 52.0% 21111111 259 236 4951.069 0.301 52.3% 21222221 55 88 143 7.615 0.006 38.5% 22111111 45 53 980.653 0.419 45.9% 11222221 44 43 87 0.011 0.915 50.6% 22111221 40 46 860.419 0.518 46.5% 11111121 38 30 68 0.941 0.332 55.9% 22111222 12 11 230.043 0.835 52.2% 21111121 3 10 13 3.769 0.052 23.1% 11111221 6 4 100.400 0.527 60.0% 21221221 1 5 6 2.667 0.102 16.7% 11221221 3 2 5 0.2000.655 60.0% 21221121 3 2 5 0.200 0.655 60.0% 11221111 0 2 2 2.000 0.1570.0% 11111222 1 1 2 0.000 1.000 50.0% TOTAL 805 805 1610

TABLE 17A analysis of 8-loocus haplotypes to two primary affectedchildren per family: “either/both sib DR3/4” subgroup. TOTAL transmittedto NOT transmitted TRANSMISSIONS McNemar chi- P-value haplotype affectedto affected OBSERVED squared statistic (1 df) % TRANSMISSION 11111111133 121 254 0.567 0.451 52.45 21111111 105 101 206 0.078 0.780 51.0%21222221 30 36 66 0.545 0.460 45.5% 22111111 22 26 48 0.333 0.564 45.8%11222221 19 19 38 0.000 1.000 50.0% 22111221 16 22 38 0.947 0.330 42.1%22111222 14 14 28 0.000 1.000 50.0% 22111222 8 6 14 0.286 0.593 57.1%21111121 1 5 6 2.667 0.102 16.7% 11111221 2 0 2 2.000 0.157 100.0% TOTAL350 350 700

TABLE 17B TDT analysis of 8-loocus haplotypes to two primary affectedchildren per family: “neither sib DR3/4” subgroup.. TOTAL transmitted toNOT transmitted TRANSMISSIONS McNemar chi- P-value haplotype affected toaffected OBSERVED squared statistic (1 df) % TRANSMISSION 11111111 138126 264 0.545 0.460 52.3% 21111111 128 112 240 1.067 0.302 53.3%21222221 22 44 66 7.333 0.007 33.3% 22111111 23 29 52 0.692 0.405 44.2%11222221 28 20 48 1.333 0.248 58.3% 22111111 21 25 46 0.348 0.555 45.7%11111121 17 11 28 1.286 0.257 60.7% 22111222 3 5 8 0.500 0.480 37.5%11111221 2 4 6 0.667 0.414 33.3% 21111121 2 4 6 0.667 0.414 33.3%11221221 3 1 4 1.000 0.317 75.0% 21221121 2 2 4 0.000 1.000 50.0%21221221 0 4 4 4.000 0.046 0.0% 11221111 0 2 2 2.000 0.157 0.0% 111112221 1 2 0.000 1.000 50.0% TOTAL 390 390 780

TABLE 18 Allele frequencies compared between Filipino controls anddiabetics. WT Allele WT allele Frequency frquency in SNP # in controlsdiabetics P-value by z-test 1 0.559 0.461 0.077 2 0.989 1.000 0.480 30.793 0.882 0.031 4 0.824 0.933 0.003 5 0.809 0.882 0.075 6 0.814 0.8820.097 7 0.723 0.770 0.362 8 1.000 1.000 1.000 n = 188 178 chromosomes

TABLE 19 Computationally estimated haplotype frequencies comparedbetween Filipino controls and diabetics. MLE MLS frequency: frequency:COUNT: COUNT: exp exp chi- controls Diabetics O.R. control diabeticsTotal count control diabetic square P-value IL4R 11111111 0.318 0.3291.1 59.7 58.5 118.2 60.7 57.5 0.033 0.855 11111121 0.074 0.074 1.0 14.013.2 27.2 14.0 13.2 0.000 0.987 11222221 0.145 0.058 0.4 27.3 10.3 37.619.3 18.3 6.727 0.009 21111111 0.390 0.441 1.2 73.3 78.5 151.8 78.0 73.80.582 0.445 21212221 0.032 0.051 1.6 6.0 9.0 15.0 7.7 7.3 0.776 0.378 n= 188 178 366

TABLE 20 Observed haplotypes and frequencies compared between Filipinocontrols and diabetics.. calc. calc. frequency: frequency: COUNT: COUNT:exp exp chi- haplotype controls Diabetics O.R. control diabetics Totalcount control diabetic square P-value IL4R x1111111 0.707 0.770 1.4133.0 137.0 270.0 138.7 131.3 0.480 0.489 x1111121 0.074 0.112 1.6 14.020.0 34.0 17.5 16.5 1.413 0.235 x1222221 0.154 0.067 0.4 29.0 12.0 41.021.1 19.9 6.155 0.013 x1212221 0.032 0.051 1.6 6.0 9.0 15.0 7.7 7.30.776 0.378 x1222111 0.005 0.000 0.0 1.0 0.0 1.0 0.5 0.5 0.947 0.331x2111111 0.011 0.000 0.0 2.0 0.0 2.0 1.0 1.0 1.894 0.169 x1221121 0.0160.000 0.0 3.0 0.0 3.0 1.5 1.5 2.840 0.092 n = 188.0 178.0 366 overall14.504 0.024

TABLE 21 Allele Frequencies in Patients and Controls Freq in SNP MajorMinor Freq in T1D Gene Description allele allele Controls patientsp-value^(a) O.R.^(b) 95% CI IL4R 5′ (−3223)^(c) C T 41.9% 51.1% 0.101.45 0.96, 2.19 5′ (−1914)^(c) C T 44.1% 42.7% 0.86 1.06 0.70, 1.60I50V^(d) A G 44.1% 53.9% 0.06 1.48 0.98, 2.23 N142N^(d) C G 1.1% 0.0%0.17 0.00 E375A^(d) A C 20.7% 11.7% 0.02 0.50 0.28, 0.90 L389L^(d) G T17.6% 6.7% 0.001 0.34 0.17, 0.67 C406R^(d) T C 19.1% 11.7% 0.05 0.560.31, 0.99 S478P^(d) T C 18.6% 11.7% 0.06 0.58 0.32, 1.04 Q551R^(d) A G27.7% 23.3% 0.34 0.80 0.50, 1.27 S761P^(d) T 100.0% 100.0% n.a. n.a. IL45′ (−524)^(e) C T 30.9% 34.4% 0.46 1.18 0.76, 1.82 IL13 5′ (−1512)^(f) AC 30.6% 41.0% 0.05 1.58 1.03, 2.42 5′ (−1112)^(f) C T 23.1% 30.9% 0.1171.49 0.94, 2.37 Intron 3^(f) C T 40.4% 45.6% 0.32 1.23 0.82, 1.86R110Q^(f) G A 39.9% 46.1% 0.23 1.29 0.85, 1.95^(a)Differences in allele frequencies between cases and controls weretested using a chi-square test.^(b)Odds ratios refer to the minority allele.^(c)Accession sequence AC004525.1;^(d)Accession sequence X52425.1;^(e)Accession sequence M23442.1;^(f)Accession sequence U10307.1

TABLE 22 Pairwise Linkage Disequilibrium Between IL4R SNPs −3223 (T)−1914 (T) 50 (G) 142 (G) 375 (C) 389 (T) 406 (C) 478 (C) 551 (G) −3223(T) — −0.183*** 0.176*** 0.004 −0.063*** −0.068*** −0.061*** −0.059***−0.068*** −1914 (T) −1.0 — −0.068*** −0.005 0.105 0.029* 0.014 0.0110.027   50 (G) 0.75 −0.81 — 0.006 −0.049** −0.067*** −0.043** −0.039**−0.079***  142 (G) 1.0 −1.0 1.0 — −0.002 −0.002 −0.002 −0.002 −0.003 375 (C) −0.70 0.70 −0.53 −1.0 — 0.139*** 0.152*** 0.147*** 0.145*** 389 (T) −0.93 0.29 −0.86 −1.0 1.0 — 0.126*** 0.122*** 0.122***  406 (C)−0.74 0.13 −0.49 −1.0 1.0 0.89 — 0.150*** 0.133***  478 (C) −0.74 0.62−0.48 −1.0 1.0 0.85 1.0 — 0.135***  551 (G) −0.56 0.17 −0.65 −1.0 0.960.96 0.96 1.0 —Top diagonal (D = P_(ab) − P_(a) · P_(b)).Bottom diagonal D′ (normalized linkage disequilibrium, D′ = D/Dmax).All values refer to the minority allele indicated in the table.Statistically significant linkage disequilibrium values are indicated asfollows:* p < 0.05;**p < 0.01;***p < 0.0001

TABLE 23 Pairwise Linkage Disequilibrium Between IL4 and IL13 SNPs IL13-IL4-524 (C) IL13-1512 (C) 1112 (T) IL13 intr (T) IL13 110 (A) IL4-524(C) — 0.062*** 0.069*** 0.024 0.063*** IL13-1512 (C) 0.29 — 0.163***0.057*** 0.058** IL13-1112 (T) 0.41 1.0 — 0.077*** 0.078*** IL13 intr(T) 0.13 0.31 0.54 — 0.201*** IL13 110 (A) 0.34 0.31 0.55 0.84 —Top diagonal (D = P_(ab) − P_(a) · P_(b)).Bottom diagonal D′ (normalized linkage disequilibrium, D′ = D/Dmax).Statistically significant linkage disequilibrium values are indicated asfollows:* p < 0.05;**p < 0.01;***p < 0.0001

TABLE 24 Genotype Frequencies in Diabetics and Controls. genotypecontrols T1DM Fisher's exact test OR 95% CI IL4R_3223 CC 29.0% 24.1%0.1584 0.78 (0.40, 1.51) CT 58.1% 51.7% 0.77 (0.43, 1.38) TT 12.9% 24.1%2.15 (0.99, 4.68) IL4R_1914 CC 31.2% 36.0% 0.6636 1.24 (0.67, 2.29) TC49.5% 42.7% 0.76 (0.43, 1.36) TT 19.4% 21.3% 1.13 (0.55, 2.33) IL4R 50AA 34.7% 23.6% 0.1846 0.58 (0.30, 1.11) AG 43.2% 44.9% 1.08 (0.60, 1.93)GG 22.1% 31.5% 1.62 (0.84, 3.13) IL4R 142 CC 97.9% 100.0% 0.4978 CG 2.1%0.0% IL4R 375 AA 62.1% 78.7% 0.038 2.25 (1.17, 4.33) AC 34.7% 19.1% 0.44(0.23, 0.87) CC 3.2% 2.2% 0.70 (0.12, 4.32) IL4R 389 GG 67.4% 87.6%0.002 3.43 (1.60, 7.37) GT 30.5% 11.2% 0.29 (0.13, 0.63) TT 2.1% 1.1%0.53 (0.05, 5.93) IL4R 406 CC 2.1% 2.2% 0.0585 1.07 (0.15, 7.76) TC33.7% 19.1% 0.46 (0.24, 0.92) TT 64.2% 78.7% 2.05 (1.06, 3.97) IL4R 478CC 2.1% 2.2% 0.0904 1.07 (0.15, 7.76) TC 32.6% 19.1% 0.49 (0.25, 0.96)TT 65.3% 78.7% 1.96 (1.01, 3.79) IL4R 551 AA 52.6% 57.3% 0.5613 1.21(0.68, 2.16) AG 38.9% 38.2% 0.97 (0.53, 1.76) GG 8.4% 4.5% 0.51 (0.15,1.76) IL4-590 CC 10.5% 12.4% 0.8143 1.20 (0.48, 2.98) CT 40.0% 42.7%1.12 (0.62, 2.01) TT 49.5% 44.9% 0.83 (0.47, 1.49) IL13INT3 C CC 33.7%31.5% 0.5413 0.90 (0.49, 1.68) CT 50.5% 46.1% 0.84 (0.47, 1.49) TT 15.8%22.5% 1.55 (0.74, 3.25) IL13 110 AA 12.6% 27.0% 0.0252 2.55 (1.19, 5.49)GA 54.7% 38.2% 0.51 (0.28, 0.92) GG 32.6% 34.8% 1.10 (0.60, 2.03)IL13_1512 AA 48.4% 34.8% 0.1363 0.57 (0.31, 1.03) AC 41.9% 48.3% 1.29(0.72, 2.32) CC 9.7% 16.9% 1.89 (0.78, 4.57) IL13_1112 CC 60.2% 51.7%0.2429 0.71 (0.39, 1.27) CT 33.3% 34.8% 1.07 (0.58, 1.97) TT 6.5% 13.5%2.26 (0.81, 6.31)

TABLE 25A Molecular IL-4 R Haplotypes in Filipino Diabetics and Controls7 SNP Frequency Frequency chi-square Haplotype in Controls in Diabeticsp-value O.R. 95% CI CAGTTAT 70.7% 77.0% 0.49 1.38 (0.9, 2.2) CCTCCGT15.4% 6.7% 0.013 0.40 (0.2, 0.8) GAGTTAT 1.1% 0.0% 0.50 0.0 (0.0, 5.8)CAGTTGT 7.4% 11.2% 0.23 1.57 (0.8, 3.2) CCGCCGT 3.2% 5.1% 0.38 1.62(0.6, 4.6) CCTTTGT 1.6% 0.0% 0.27 0.0 (0.0, 3.5) CCTGTAT 0.5% 0.0% 0.980.0 (0.0, 15.8) total (6 d.f.) 0.076 n = 188 178

TABLE 25B Estimated IL4R 10-SNP Haplotype Frequencies in Diabetics andControls IL4R 10 Locus Controls^(a) TIDM P- 8 locus H-ID Haplotype N= 188 std dev N = 180 std dev O.R. 95% CI value^(b) H-5A CCACGTCCGT 5.8%(1.7%) 0.0% 0.00 (0.0, 0.8) 0.001 H-5B CTACCTCCGT 8.5% (2.3%) 5.8%(2.2%) 0.66 (0.3, 1.6) 0.33 H-3A CTGCCTCCGT 0.9% (0.6%) 0.0% 0.00 (0.0,6.0) 0.21 H-3B TCGCCTCCGT 0.0% 0.9% (1.1%) n. a. 0.19 Total (23 d. f.)0.04^(a) Maximum likelihood haplotype frequencies were computed using anExpectation-Maximization (EM) algorithm (see Excoffier and Slatkin 1995)as implemented by the Arlequin software program L. Excoffier, Universityof Geneva, CH). The standard deviation was computed carrying out 100boostrap replicates. 24 of the possible 512 haplotypes were observed.The four shown here all include the disease associated seven SNPhaplotype. The two 8 SNP haplotypes containing the seven SNP haplotypewere#designated H-3 and H-5 in reference Mirel et al, (in press) Thestandard deviation was computed carrying out 100 boostrap replicates.^(b) Differences in allele frequencies between cases and controls weretested using a chi-square test

TABLE 26 Estimated IL4 and IL13 5-SNP Haplotype Frequencies Diabeticsand Controls 5 locus Controls TIDM Odds haploytpe N = 188 Sd N = 176* SdRatio P-value CACCA 4.1% (1.3%) 3.5% (1.7%) 0.84 CACCG 8.9% (2.2%) 10.6%(2.9%) 1.21 CACTA 1.3% (1.1%) 3.0% (1.8%) 2.26 CATTA 0.0% 0.6% (0.5%) n.a. CCCCG 1.5% (1.0%) 0.9% (0.9%) 0.61 CCTCG 1.1% (0.9%) 5.5% (2.3%) 5.190.02 CCTTA 12.6% (2.4%) 9.3% (2.0%) 0.71 CCTTG 0.0% 0.4% (0.7%) n. a.TACCG 33.6% (3.8%) 21.9% (3.9%) 0.55 0.03 TACTA 16.4% (3.3%) 16.3%(3.0%) 0.99 TACTG 5.0% (1.7%) 3.2% (1.4%) 0.62 TCCCG 4.6% (1.8%) 9.8%(2.9%) 2.23 0.06 TCCTA 1.3% (1.1%) 0.0% 0.00 TCCTG 0.2% (0.5%) 0.0% 0.00TCTCG 5.3% (2.4% 1.7% (1.2%) 0.31 0.07 TCTTA 4.1% (2.1%) 12.8% (2.2%)3.47 0.004 TCTCA 0.0% 0.6% (0.7%) n. a. Total (16 d. f.) 0.005* due to missing genotypes.(order of the SNPs: IL4-524, IL13-1512, IL13-1112, IL13INT3, IL13R110)

TABLE 27 Test For Independence Between Genotype Frequencies at IL4R SNPsand Genotype Frequencies at Five IL4 and IL13 SNPs. Chromsome 5 SNPcontrols T1D IL4-524 0.15 0.001 IL13-1512 0.77 0.78 IL13-1112 0.93 0.73IL13 110 0.99 0.91 IL3 intron 3 0.99 0.019

TABLE 28 Epistasis Between IL4R and Five IL4 and IL13 SNPs Walds Nominalβ₃. Std. Error Odds ratio Chi-sq p-value IL4-524:il4r −0.22 0.10 0.814.55 0.033 IL13-1512:il4r 0.04 0.15 1.04 0.06 0.811 IL13-1112:il4r 0.100.16 1.10 0.37 0.545 IL13 110:il4r 0.14 0.09 1.15 2.24 0.135IL13INT3:il4r 0.17 0.09 1.18 3.19 0.074The Overall p-Value For All Five Tests By Permutation Analysis Was p <0.045 (see text).

TABLE 29 Probes For IL4 and IL13 Seq ID Conc. SNP Allele Probe ProbeSequence No: (μM) IL4P C582T C KW66 AACATTGTCCCCCAGTGC 63 1.2 IL4P C582TT KW89 AGCACTGGGGAACAATGTTC 64 0.9 IL13 Int 3 C DBM0161PTTCTACTCACGTGCTGACCT 65 1.0 IL13 Int 3 T DBM0164P GGTCAGCACATGAGTAGAACG66 0.3 IL13 Ex 4 R110Q DBM0136P TCAGTTGAACCGTCCCTCG 67 1.0 IL13 Ex 4R110Q DBM0181P GAGGGACAGTTCAACTGAAAC 68 1.0

TABLE 30 Amplicon Primers and Lengths Amplicon Forward Seq ID Seq IDExon Size Primer No Sequence Reverse Primer No Sequence IL4P 107 RR169B69 ACTAGGCCTCACCTGATACGA RR170B 72 CATAGAGGCAGAATAACAGGCAGA IL 13 in 3118 DBM0165B 70 CTCGGACATGCAAGCTGGAA DBM0166B 73 ACTGAATGAGACAGTCCCTGGAIL 13 ex 4 187 DBM0167B 71 AATCGAGGTGGCCCAGTTTGTA DBM0168B 74CCTAACCCTCCTTCCCGCCTA

TABLE 31 Amplicon Primers and Lengths for IL4R Amplicon Seq ID Exon SizeAllele Primer No Sequence T(−1914)C 49 T DBM0659 75ACTGACTTATCTTTACTGTCACTTCT DBM0661 76 GCAAGACAGCCACCAACCC T(−1914)C 49 CDBM0660 77 TGACTTATCTTTACTGTCACTTCC DBM0661 76 GCAAGACAGCCACCAACCCC(−3223)T 42 C DBM0672 79 CCTGCTCCCAGGACTGAC DBM0667 80CCCAGACTTTATCTGTGACTGCTC C(−3223)T 42 T DBMO671 81 CCTGCTCCCAGGACTGATDBM0667 80 CCCAGACTTTATCTGTGACTGCTC

TABLE 32 Amplicon Primers and Lengths for IL13 Amplicon Seq ID Exon SizeAllele Primer No Sequence A(−1512)C 44 A DBM0650 82 GGAAACAGGCCCGTAGADBM0652 83 GAGTGCCGCTACTTGGCC A(−1512)C 43 C DBM0651 84 GAAACAGGCCCGTAGCDBM0652 83 GAGTGCCGCTACTTGGCC C(−1112)T 55 C DBM0656 85TCTGGAGGACTTCTAGGAAAAC DBM0658 86 TGCAGCCATGTCGCCTTT C(−1112)T 55 TDBM0657 87 TCTGGAGGACTTCTAGGAAAAT DBM0658 86 TGCAGCCATGTCGCCTTT

1. A method for determining an individual's risk for type 1 diabetescomprising detecting the presence of at least one type 1diabetes-associated IL4R, IL4, or IL13 polymorphism in a nucleic acidsample of the individual, wherein the presence of said polymorphismindicates the individual's risk for type 1 diabetes.
 2. The method ofclaim 1, further comprising detecting at least one other polymorphismand wherein said polymorphisms comprise a haplotype.
 3. The method ofclaim 1, wherein the nucleic acid sample comprises DNA.
 4. The method ofclaim 1, wherein the nucleic acid sample comprises RNA.
 5. The method ofclaim 1, wherein the at least one polymorphism is detected byamplification.
 6. The method of claim 5, wherein the at least onepolymorphism is detected by a polymerase chain reaction.
 7. The methodof claim 1, wherein the at least one polymorphism is detected bysequencing.
 8. The method of claim 1, wherein the at least onepolymorphism is detected by amplifying a target region containing the atleast one polymorphism; hybridization of at least one sequence-specificoligonucleotides that hybridizes under stringent conditions to the atleast one polymorphism; and detecting the hybridization.
 9. The methodof claim 1, wherein the at least one polymorphism is selected from thelist of polymorphisms listed in Table
 2. 10. The method of claim 1,wherein the at least one polymorphism is selected from the list ofpolymorphisms listed in Table
 21. 11. The method of claim 10, whereinthe at least one polymorphism is a combination of two or morepolymorphisms selected from the polymorphisms listed in Table
 21. 12.The method of claim 1, wherein the at least one polymorphism is an IL4Rpolymorphism.
 13. The method of claim 12, further comprising detectingat least one IL13 polymorphism.
 14. The method of claim 12, furthercomprising detecting at least one IL4 polymorphism.
 15. A kit fordetermining an individual's risk for type 1 diabetes, where the kitcomprises at least one sequence-specific oligonucleotide that comprisesa sequence that hybridizes under stringent condition to at least onetype 1 diabetes-associated IL4R, IL4, or IL13 polymorphism.
 16. The kitof claim 15, further comprising reagents for the amplification of atarget region, wherein said target region comprises the at least onepolymorphism.
 17. The kit of claim 16, wherein said reagents foramplification comprise at least one primer.
 18. The kit of claim 15,wherein said at least one sequence-specific oligonucleotide is labeled.19. The kit of claim 15, further comprising a reagent to detect thelabel.
 20. The kit of claim 15, wherein the at least one polymorphism isselected from the list of polymorphisms set forth in Table 21.